CN106526017A - Ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrometry screening method for nutrition enhancer in milk and dairy product - Google Patents
Ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrometry screening method for nutrition enhancer in milk and dairy product Download PDFInfo
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Abstract
Belonging to the technical field of food safety detection, the invention discloses an ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrometry screening method for a nutrition enhancer in milk and dairy product. The method includes: utilizing optimized QuEChERS method to perform pretreatment on a sample; using ultra-high performance liquid chromatography-quadrupole electrostatic field orbit ion trap mass spectrometry for screening, adopting full scanning/data dependent acquisition, full scanning/variable data non-dependent acquisition, and full scanning/all-ion fragmentation acquisition scanning modes, selecting positive ion or negative ion mode scanning to obtain complete first-order and second-order spectrum; applying Exact Finder 2.5 software to process the obtained first-order and second-order spectrum, and extracting compound information; and comparing the information in the established spectrum library with the screening result of the detected object, and confirming the composition of the nutrition enhancer in the milk and dairy product. The mass spectrometer used in the invention has the main advantages of high resolution, accurate qualitative and quantitative result, high sensitivity and high quality precision, etc., and can be applied to analysis of complex matrices.
Description
【Technical field】
The invention belongs to technical field of food safety detection, in more particularly to a kind of milk and milk productses, nutrition enhancer is super
High performance liquid chromatography-level Four bar electrostatic field orbit ion trap mass spectrum screening method.
【Background technology】
What nutrition enhancer was referred to strengthen nutritional labeling and added the natural or synthetic in food belongs to day
The material of right nutrient scope, it is referred to as condensed food through the food of intensive treatment.China specifies the nutrition enhancer for allowing to use
Including the species that GB14880 and GB2760 specify.Nutrition enhancer is broadly divided into vitaminss, discord saturated fat acids, nucleoside
With ucleotides.Add a certain amount of nutrition enhancer in milk and milk productses, can balanced nutrients composition, make up nutrient lack
Lose.In recent years, people were increasingly paid close attention to for the safety problem of milk and milk productses, and exogenous specified risk material is to affect milk and milk productses
One importance of safety is increasingly taken seriously.Nutrition enhancer is a kind of conventional food additive in milk product, according to《GB
14880-2012 food enrichments use standard》Announce with 5 supplements, addition vitamin, mineral, nucleoside and nucleotide,
The nutrition enhancer such as unsaturated fatty acid, play a part of balanced nutrients composition with make up nutrient disappearance.Nutrition enhancer is made
For a kind of important food additive, have become milk and milk productses industrial technological advancement and scientific and technical innovation important impetus it
One.However, if reinforcing dosage is inappropriate, body nutritive equilibrium can be caused to be destroyed, or even because hyperphagia causes poisoning.
Therefore, it is necessary to the usage amount of nutrition enhancer is controlled and is monitored.However, currently used for milk and milk productses
The high flux of exogenous specified risk material, method for quick are less, pre-treatment complex operation, and material is high with equipment cost, only
It is limited to several or a class compound quick detection.In recent years, the application of QuEChERS wide spectrums extracting method and by instrument join
Developed rapidly with milk and milk productses exogenous specified risk material Screening analysis technology is caused.Binding isotherm knowledge of the present invention and reality
Situation, establishes combined gas chromatography mass spectrometry high flux, the rapid screening analysis method of sweet nutrition enhancer in milk and milk productses.
【The content of the invention】
The present invention is directed to above-mentioned present situation, there is provided and the Ultra Performance Liquid Chromatography of nutrition enhancer in a kind of milk and milk productses-
Level Four bar electrostatic field orbit ion trap mass spectrum screening method, the main advantage of the method is high resolution, qualitative and quantitative result
Accurately, 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 nutrition enhancer-level Four bar electrostatic field orbit ion trap mass spectrum in a kind of milk and milk productses
Screening 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 fortification in measured object sample
Immunomodulator compounds information；
4) compound by the fortification immunomodulator compounds information in nutrition enhancer standard spectrum storehouse with measured object sample is believed
Breath is compared, and carries out final decision structure with battalion in quantitative milk and milk productses by the chromatographic mass spectrometry information of analytical standard material
The composition of foster hardening agent.
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 as to 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 nutrition enhancer standard substance；
2) with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph to nutrition enhancer standard substance
Standard solution carries out examination, using electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF sweep
Pattern is retouched, the complete one-level of standard solution and second order mses figure is obtained；
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 nutrition enhancer.
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-18 aQ pre-columns, 10mm × 2.1mm, 1.9 μm, Hypersil Gold aQ C-18
Post, 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 formic acid
The aqueous solution of ammonium, 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；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 target
Value 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 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
Nutrition enhancer 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 nutrition enhancer in quantitative milk and milk productses.The screening technique of the present invention, can be with
Disposable quantitative, qualitative detection many kinds of substance (23 kinds of nutrition enhancer), 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 compound
Under 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 Mass accuracy not under
Drop；Sensitivity is high, detects the frequency of ion cyclotron resonance, and does not need extra detector；Wide dynamic range, up to five orders of magnitude；
With quick positive and negative switched scan pattern, analysis time is greatly saved, improves analysis throughput；Stability is high, only needs correction one in a week
It is secondary；Quantitation capabilities are high, can compare with triple quadrupole bar；Mass range is wide, can carry out polypeptide analysis；In addition, Q-Orbitrap
Instrumentation it is easy, operating cost is low, can be applicable to the analysis of complex matrices.As a result show：23 kinds of nutrition enhancer are linear
In the range of there is good linear relationship, correlation coefficient is all higher than 0.99, high (4 × CC β), in (2 × CC β), low (1 × CC β) three
Under individual pitch-based sphere, the recovery of standard addition of substrate reaches 82%-112%, repeats experiment 6 times, and its relative standard deviation is 0.3%-
6.3%.It is determined that limit (CC α) and measurement capacity (CC β) are respectively 0.1 μ g/kg-1.91 μ g/kg and 0.17 μ g/kg-3.18 μ g/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 is level Four bar-orbit ion trap mass spectrograph Full MS/vDIA scan pattern figures.
【Specific embodiment】
The invention provides in a kind of milk and milk productses nutrition enhancer Ultra Performance Liquid Chromatography-level Four bar electrostatic field rail
Road ion trap mass spectrometry screening method, it is characterised in that：Equipment used in the method includes that Ultra Performance Liquid Chromatography-level Four bar is quiet
Electric 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 nutrition enhancer spectrum storehouse is compared, and flows including extracting
The accurate matter at the retention time of chromatograph, molecular ion peak and its fragmentation of ions peak abundance ratio, molecular ion peak and fragmentation of ions peak
Amount number, molecular ion peak and isotope ion peak abundance ratio, confirm the composition of nutrition enhancer 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, is furnished with electric spray ion source.
(1) chromatographic condition
Chromatographic column：Accucore C-18 aQ 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℃；Mobile phase：A is 0.1% (v/v) formic acid and 4mmol/L formic acid aqueous ammoniums, is flowed
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
Measure 5 μ L；Collision gas：High-purity argon gas (purity >=99.999%).Electro-spray ionization (ESI) ion source：Single holotype is negative
Pattern；Sheath gas：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℃；Capillary temperature：320℃.
(2) Mass Spectrometry Conditions
Using Full MS/vDIA scan patterns.First mass spectrometric scanning resolution is 70000FWHM；Automatic growth control target
Value 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 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 the information such as the ionization pattern and retention time of 23 kinds of nutrition enhancer standard substances；Table 2 lists 23
Plant nutrition enhancer standard database partial content.
1 23 kinds of nutrition enhancer standard substance information of table
2 23 kinds of nutrition enhancer 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 nutrition enhancer standard substance 10mg (being accurate to 0.1mg) is weighed respectively, is placed in 10mL in volumetric flask, according to
According to standard substance, in different solvents (methanol, acetonitrile and toluene), dissolubility is needed with determining, and is dissolved from suitable solvent, fixed
Hold to 10mL, be configured to single compound standard reserving solution, keep in dark place in -20 DEG C.Pipette single compound standard reserving solution
In 100mL volumetric flasks, with methanol-water solution (1:1, v/v) scale is diluted and be settled to, the standard substance of respective concentration is prepared
Hybrid 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 dry up instrument be blown to it is near dry, be separately added into the 10 μ L of nutrition enhancer 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 are in volumetric flask, each to add
Methanol 5mL, dilutes and is settled to scale with 8mmol/L formic acid aqueous ammoniums, be 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
Solution.Matrix matching standard solution answers matching while using.
3. the pre-treatment of sample
The milk power for infant and young children 1.5g (being accurate to 0.01g) for having dissolved is weighed in 50mL tool plug polystyrene centrifuge tubes
In, the water of 45 DEG C ± 5 DEG C of 10mL is added, being vortexed fully to mix is completely dispersed sample, cools down in being put into circulating water.Add
10mL 1% (v/v) acetic acidacetonitrile/water (84:16, v/v) mixed solution, vortex mixed 30s.Addition 6.0g anhydrous magnesium sulfates,
Ceramic homogenizing stone, 1.45g anhydrous sodium acetates, vortex 30s, mechanical shaking extraction 1min.Centrifuge tube is placed in into centrifuge, temperature is set as
, 5min is centrifuged with 10000r/min, 200 μ L filtrates are taken in 2mL sample introduction bottles, add 500 μ L 8mmol/L ammonium formate water by 4 DEG C
Solution and 300 μ L methanol.It is measured with Ultra Performance Liquid Chromatography-level Four bar-orbit ion trap mass spectrum.
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 changes for belonging to class in storehouse with the nutrition enhancer standard information set up
Compound dissociation pathways are compared, and judge the molecular formula and structural formula of the compound, by the chromatographic mass spectrometry information of analytical standard material
Carry out it is final judge with it is quantitative.
(2) Method validation
The standard curve of a methods, the range of linearity and correlation coefficient
23 kinds of nutrition enhancer series hybrid standard substance solutions are prepared, concentration is 0.1 μ g/kg, 0.5 μ g/kg, 1 μ g/
kg、2μg/kg、4μg/kg、8μg/kg、10μg/kg、20μg/kg、40μg/kg、80μg/kg、100μg/kg、200μg/kg、400
μg/kg、500μg/kg.As a result show, in detection method 23 kinds of nutrition enhancer in its corresponding concentration range, correlation coefficient
(r2) 0.99 is all higher than, in good linear relationship.
The lower limit of quantitation of b 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 0.1 μ g/kg-1.91 μ g/kg and 0.17 μ g/kg-3.18 μ g/kg.
Precision is obtained by the relative standard deviation for counting 6 parallel laboratory test response rate results, and determinand is extracted net
After change, with level Four bar-orbit ion trap Mass Spectrometer Method, the average recovery rate for calculating 23 kinds of nutrition enhancer is inclined with relative standard
Difference.The response rate and relative standard deviation result are respectively 82%-112% and 0.3%-6.3%, the method accuracy set up and
Precision is good.Table 3 below lists the methodology parameter of nutrition enhancer in pasteurization milk.
The range of linearity of nutrition enhancer in 3 milk power for infant and young children of table, determine limit, measurement capacity, correlation coefficient, averagely
The response rate and precision (n=6)
The examination object of the present invention is 196 batch pasteurization milks, instantaneous ultrahigh-temperature sterilization breast, beverage containing milk, fermentations
Breast, dilute cream and milk power for infant and young children.
Fig. 1 is variable data non-dependent collection Variable Data Independent Acquisition (vDIA),
It is improved on the basis of non-dependent drainage pattern, according to the mass charge ratio range is distributed by target substance, adjusts each " window
The size of mouth ", while meeting the requirement of selectivity and scanning speed.One circulation includes the broken of 1 full scan and 20 passages
Piece is scanned, and because in material to be analyzed more than 95% mass-to-charge ratio is distributed in 87.0-500.0, first vDIA scans through information
Mass-to-charge ratio 87.80116-512.97169 is set as following 16 passages by list：112.80116、137.81253、
162.82390、187.83527、212.84663、237.85800、262.86937、287.88074、312.89211、
337.90348、362.91485、387.92622、412.93758、437.94895、462.96032、487.97169.Isolation window
Mouth scope (Isolation Window) is set to 25.0Da, and 112.80116 sweep limitss are mass-to-charge ratio 87.80116-
137.80116(112.80116±25.0).Other 5% compound mass-to-charge ratioes to be analyzed are distributed in 500.0-900.0, second
VDIA scans through information list and mass-to-charge ratio 500.50011-900.97169 is set as following 4 passages：550.50011、
650.54559th, 750.59106,850.63654, isolation window ranges (IsolationWindow) is set to 100.0Da.Full
What MS/Variable Data Independent Acquisition were scanned is in the nature the part-time of full scan for broken
Segment scan is split, 1.8s completes single pass circulation, and 1 circulation obtains a molecular ion and sweeps full figure, and 20 fragmentation fragments are swept
Information in Mass Spectra is not lost in tracing, this pattern, and full scan and two grades of fragmentation fragments are respectively formed profile diagram for qualitative and quantitative, and believe
Breath list need not be adjusted according to analysans, i.e., Mass Spectrometry Conditions are consistent, and sample can be used Jing after single injected sampling measure and be divided
Analysis software is repeatedly analyzed for different analysans, and data are easily traced to the source, and have more preferable data integrity and flux.
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, nutrition enhancer standard in the milk and milk productses set up is chosen
Data base, retention time extract window and are set as retention time ± 3 times shift of retention time standard deviation.Extraction chromatography peak matter lotus
It is 3ppm than allowing biggest quality deviation setting.Window is extracted when the retention time with setting and mass-to-charge ratio mass deviation to extract
It is more than or equal to 8.3 × 10 to response intensity4During chromatographic peak, sample is tentatively judged as positive, carries out next step confirmation work,
If not extracting chromatographic peak under these conditions, preliminary judgement sample is feminine gender.Tentatively it is judged as that the sample of feminine gender is carried out
The extraction and analysis of feature fracture fragment, carries out Screening analysis to unknown object material, and then reaches a conclusion.Confirmation work is main
Carried out by two aspects of isotope ion peak information and two grades of fragmentation fragment abundance ratios, and solve isomerss chromatograph altogether
The confirmation difficult point of outflow, by with M+1 features13C, and with M+2 features34S、81Br、37The isotope ion peak abundance of Cl
Than being applied to confirm step.
At present for the research of fortification agent high flux rapid screening technology in milk and milk productses is still blank.For breast
And the high flux of nutrition enhancer residual, method for quick are less in milk product, pre-treatment complex operation, material and equipment into
This height, is only limitted to several or a class compound quick detection, remains often special according to the structure of compound during quick analysis more
Point is detected after compound is grouped.In recent years, the application of QuEChERS wide spectrums extracting method and by Instrument crosslinking cause breast and
Milk product exogenous specified risk material Screening analysis technology is developed rapidly.Herein for above-mentioned present situation, with high resolution mass spectrum four
Level 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, establishes 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 examination of nutrition enhancer point in the milk and milk productses of foundation
Analysis method is examined, and in all storehouses, potential target compound has good linear relationship, correlation coefficient in linear scope
0.99, under high, medium and low three pitch-based spheres is all higher than, the response rate reaches 82%-112%, repeats experiment 6 times, its relative standard
Deviation is 0.3%-6.3%.It is determined that limit (CC α) and measurement capacity (CC β) are respectively 0.1 μ g/kg-1.91 μ g/kg and 0.17 μ g/
Kg-3.18 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 compared with national standard method with degree of accuracy.
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, the Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum of nutrition enhancer is sieved
Checking 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 nutrition enhancer in measured object sample
Compound information；
4) the compound information by the fortification immunomodulator compounds information in nutrition enhancer standard spectrum storehouse with measured object sample is entered
Row is compared, and carries out final decision structure by the chromatographic mass spectrometry information of analytical standard material strong with nutrition in quantitative milk and milk productses
The composition of agent.
2. in a kind of milk and milk productses according to claim 1, the Ultra Performance Liquid Chromatography-level Four bar of nutrition enhancer is quiet
Electric 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
In plug polystyrene centrifuge tube, 45 DEG C ± 5 DEG C of water is added, being vortexed fully to mix is completely dispersed sample, is put in circulating water
Cooling；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 are 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, the Ultra Performance Liquid Chromatography-level Four bar of nutrition enhancer is quiet
Electric field orbit ion trap mass spectrum screening method, it is characterised in that：Step 2) it is specially：By Ultra Performance Liquid Chromatography-level Four bar
The mass spectrographic full scan pattern of electrostatic 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 nutrition enhancer Ultra Performance Liquid Chromatography-level Four bar
Electrostatic field orbit ion trap mass spectrum screening method, it is characterised in that：Step 3) it is specially：By analyzing what second order mses figure was obtained
Fragmentation fragment data, tentatively judges the material belonging kinds, and then other piece segment informations of the retention time chromatographic peak are carried out
Analysis, including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, so as to be inferred to the structure of matter and elementary composition.
5. in a kind of milk and milk productses according to claim 1, the Ultra Performance Liquid Chromatography-level Four bar of nutrition enhancer is quiet
Electric 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 nutrition enhancer standard substance；
2) with Ultra Performance Liquid Chromatography-standard of the level Four bar electrostatic field orbit ion trap mass spectrograph to nutrition enhancer standard substance
Solution carries out examination, using electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scanning mould
Formula, 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 nutrition
The standard spectrum storehouse of hardening agent.
6. in a kind of milk and milk productses according to claim 1, the Ultra Performance Liquid Chromatography-level Four bar of nutrition enhancer is quiet
Electric 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 matter
The chromatographic condition of spectrometer 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℃.
7. in a kind of milk and milk productses according to claim 1, the Ultra Performance Liquid Chromatography-level Four bar of nutrition enhancer is quiet
Electric 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 matter
The Mass Spectrometry Conditions of spectrometer 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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| CN112881554A (en) * | 2021-01-15 | 2021-06-01 | 陕西科技大学 | Detection method for nicotine drug chloride and metabolite thereof in mutton |
| CN113030296A (en) * | 2021-02-09 | 2021-06-25 | 上海市食品药品检验研究院 | Method for screening abamectin and emamectin benzoate degradation products in honeysuckle and planting soil thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108008041A (en) * | 2017-12-06 | 2018-05-08 | 广州市食品检验所 | The detection method of residual quantity of acrylamide monomer in food grade polyacrylamide |
| CN109813813A (en) * | 2019-01-18 | 2019-05-28 | 中国农业科学院农业质量标准与检测技术研究所 | Identify the method for ultra-high-temperature sterilized milk and reconstituted milk based on lipid group |
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| CN112881554A (en) * | 2021-01-15 | 2021-06-01 | 陕西科技大学 | Detection method for nicotine drug chloride and metabolite thereof in mutton |
| CN113030296A (en) * | 2021-02-09 | 2021-06-25 | 上海市食品药品检验研究院 | Method for screening abamectin and emamectin benzoate degradation products in honeysuckle and planting soil thereof |
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Application publication date: 20170322 |