CN106526019A - Method for using ultra-high performance liquid chromatography-quadrupole electrostatic field ion trap mass spectrum to screen vitamin and natural antioxidant in health care product - Google Patents
Method for using ultra-high performance liquid chromatography-quadrupole electrostatic field ion trap mass spectrum to screen vitamin and natural antioxidant in health care product Download PDFInfo
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- CN106526019A CN106526019A CN201610967226.8A CN201610967226A CN106526019A CN 106526019 A CN106526019 A CN 106526019A CN 201610967226 A CN201610967226 A CN 201610967226A CN 106526019 A CN106526019 A CN 106526019A
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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
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention discloses a method for using an ultra-high performance liquid chromatography-quadrupole electrostatic field ion trap mass spectrum to screen vitamin and natural antioxidant in a health care product, and belongs to the technical field of food safety inspection. The method comprises the steps of firstly conducting extraction and purification on a sample using an optimized QuEChERS method; secondly conducting screening through a Q-Orbitrap, adopting three scan patterns of electrospray ion source and full scan/data dependence acquisition, full scan/variable data independence acquisition, full scan/full ion fragmentation acquisition, and obtaining integrated primary mass spectrum and secondary mass spectrum ; then adopting Exact Finder 2.5 software to conduct treatment on the obtained primary mass spectrum and secondary mass spectrum to extract information of a compound. By comparing information in an established spectrum library and information of screening results of the detected, constitution of vitamin and natural antioxidant in a green tea health care product is confirmed. The method for using the ultra-high performance liquid chromatography-quadrupole electrostatic field ion trap mass spectrum to screen vitamin and natural antioxidant in the health care product has the advantages that resolution is high, qualitative and quantitative results are accurate, sensitivity is high, quality accuracy is high and the like, and can be applicable to analysis of a complex substrate. The screening method has a very good application prospect in food industry.
Description
【Technical field】
The invention belongs to technical field of food safety detection, vitamin and natural anti-oxidation in more particularly to a kind of health product
The Ultra Performance Liquid Chromatography of agent-level Four bar electrostatic field orbit ion trap mass spectrum screening method.
【Background technology】
In the U.S., green tea is used as dietary supplement, as which contains abundant bioactive substance, including caffeine, ammonia
Base acid, vitamin etc., so that reach the effect for promoting health.Green tea extract, such as vitamin and the Natural antioxidant etc.,
Jing is commonly used for preventing and treating malnutrition, becomes thin, and reduces cholesterol levels, can also protect the skin from the wound of sunlight
Evil.Green tea extract is generally in the form of a tablet or capsule as nutrient and healthcare products selling.At present, nutrient and healthcare products become
A part for diet, to make up the sickness rate for reducing disease because of malnutrition caused by life style, so that improve the mankind
Life expectancy.However, only reasonably edible green tea extract health product can just play a part of to promote health, excessively
It is edible then certain injury can be caused to human body.Therefore, supervision department is required to vitamin in health product and the Natural antioxidant
Quantitative analyses are carried out, accurately to mark the content of vitamin and the Natural antioxidant in health food.
High flux, method for quick currently used for health product exogenous specified risk material is less, and pre-treatment operation is multiple
Miscellaneous, material is high with equipment cost, and is only limitted to several or a class compound quick detection.Vitamin is had with the Natural antioxidant
There are different structures and chemical property, while it is relatively difficult to test and analyze both materials.Therefore, health product exogenous are developed
The high flux of specified risk material, rapid screening analysis method are into development trend.
【The content of the invention】
The present invention is directed to above-mentioned present situation, there is provided the ultra high efficiency liquid phase of vitamin and the Natural antioxidant in a kind of health product
Chromatograph-level Four bar electrostatic field orbit ion trap mass spectrum screening method, the main advantage of the method is high resolution, qualitative and quantitative
As a 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：
Ultra Performance Liquid Chromatography-level Four bar electrostatic field the orbiting ions of vitamin and the Natural antioxidant in a kind of health product
Trap mass spectrum screening method, comprises the following steps：
1) the measured object sample of health product is extracted and is purified using QuEChERS methods, obtained measured object sample
Test solution；
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, extract in measured object sample vitamin and
Natural anti-oxidation immunomodulator compounds information；
4) by the vitamin of the compound information in vitamin and Natural antioxidant standard spectrum storehouse and measured object sample with
Natural anti-oxidation immunomodulator compounds information is compared, and carries out final decision structure by the chromatographic mass spectrometry information of analytical standard material
With quantitative food vitamins and the composition of the Natural antioxidant.
As a further improvement on the present invention, described health product are green tea extract health product.
As a further improvement on the present invention, step 1) it is specially：Green tea extract tablet or capsule are weighed in centrifuge tube
In, 45 DEG C ± 5 DEG C of water is added, being vortexed fully to mix is completely dispersed sample, cools down in being put into circulating water；Add volume integral
Acetic acidacetonitrile/the water mixed solution of number 1%, vortex mixed；Add anhydrous magnesium sulfate, ceramic homogenizing stone, anhydrous sodium acetate, whirlpool
Rotation, mechanical shaking extraction；After finally be centrifuged centrifuge tube, filtrate is taken in sample introduction bottle, add formic acid aqueous ammonium and methanol.
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 vitamin and Natural antioxidant standard spectrum storehouse be according to
What lower step was set up：
1) standard solution of vitamin and Natural antioxidant standard substance is prepared respectively；
2) with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph to vitamin and natural anti-oxidation
The standard solution of agent standard substance 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
Standard spectrum storehouse.
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 that 0.1% (v/v) formic acid is water-soluble with 4mmol/L ammonium formates
Liquid, Mobile phase B are 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 size 5-10 μ L；Collision gas：High-purity argon gas；
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 green tea extract health product sample is extracted and purified, and then adopts
In Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrography detection green tea extract health product vitamin with it is natural
Antioxidant, compares finally by standard spectrum storehouse, final to judge compound structure and quantitative green tea extract health product
Vitamin and the composition of the Natural antioxidant.The screening technique of the present invention, can disposable quantitative, qualitative detection many kinds of substance
(52 kinds of vitamin and the Natural antioxidant), and there is simple to operate, high precision, high resolution, qualitative and quantitative result standard
Really, the advantage such as sensitivity height, Mass accuracy height, can be applicable to the analysis of complex matrices.Especially, Q-Orbitrap (ultra high efficiencies
Liquid chromatograph-level Four bar electrostatic field orbit ion trap mass spectrograph) high resolution, in the case where trace compound sensitivity is guaranteed, point
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 does not decline；Sensitivity
Height, detects the frequency of ion cyclotron resonance, and does not need extra detector；Wide dynamic range, up to five orders of magnitude；With fast
The positive and negative switched scan pattern of speed, is greatly saved analysis time, improves analysis throughput；Stability is high, only needs correction in a week once；It is fixed
Amount ability is high, can compare with triple quadrupole bar；Mass range is wide, can carry out polypeptide analysis；In addition, Q-Orbitrap instruments
Easy to operate, operating cost is low, can be applicable to the analysis of complex matrices.As a result show：52 kinds of vitamin and the Natural antioxidant
There is good linear relationship, correlation coefficient is all higher than 0.99 in the linear scope, high (4 × CC β), in (2 × CC β), low by (1
× CC β) under three pitch-based spheres, the recovery of standard addition of substrate reaches 85%-113%, repeats experiment 6 times, and its relative standard is inclined
Difference is 0.3%-6.4%.It is determined that limit (CC α) and measurement capacity (CC β) are respectively 0.05 μ g/kg-1.91 μ g/kg and 0.08 μ g/
kg-3.18μg/kg。
Green tea extract health product sample is extracted and purified using the QuEChERS methods after optimization, it is simple fast
Victory, efficiency high can effectively exclude wherein interference of the impurity to subsequent detection.
【Description of the drawings】
Fig. 1 is that the pH of the present invention affects response surface figure with extraction liquor capacity to the response rate；
Fig. 2 is isomerss ursolic acid (Ursolic acid) and oleanolic acid (Oleanic acid) full scan and two
Level fragmentation fragment comparison chart (spiked levels 50mg kg-1)。
【Specific embodiment】
The invention provides in a kind of green tea extract health product vitamin and the Natural antioxidant ultra high efficiency liquid phase color
Spectrum-level Four bar electrostatic field orbit ion trap mass spectrum screening method, the equipment used in the method include Ultra Performance Liquid Chromatography-four
Level bar electrostatic field orbit ion trap mass spectrograph.Comprise the following steps：
First, sample is extracted and is purified using the QuEChERS methods for having optimized；
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 for having been set up information that vitamin and the Natural antioxidant composed in storehouse with measured object is compared,
Including the retention time for extracting flow chromatography, molecular ion peak and its fragmentation of ions peak abundance ratio, molecular ion peak and fragmentation of ions
The accurate mass number at peak, molecular ion peak and isotope ion peak abundance ratio, in confirmation green tea extract health product vitamin with
The composition of the Natural antioxidant.
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-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 52 kinds of standard database partial contents.
Ultra Performance Liquid Chromatography/level Four the bar of 1 52 kinds of vitamin of table and the Natural antioxidant-orbit ion trap mass spectrum ginseng
Number
2. solution is prepared
(1) preparation of standard solution
Weigh appropriate vitamin and Natural antioxidant standard substance 10mg (being accurate to 0.1mg) respectively, be placed in 10mL in
In volumetric flask, establishing criteria material dissolubility in different solvents (methanol, acetonitrile and toluene) is needed with determining, from suitable
Solvent dissolves, and 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 100mL volumetric flasks, with methanol-water solution (1:1, v/v) scale is diluted and be settled to, respective concentration is prepared
Standard substance 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
12 parts of each 5mL green tea extracts health product blank sample (determining before experiment) extracting solution are pipetted in 10mL volumetric flasks
In, instrument is dried up with vacuum nitrogen be blown to closely do, the vitamin and Natural antioxidant standard substance for being separately added into 1000 μ g/L is mixed
Close 10 μ L of solution, 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
It is in volumetric flask, each to add methanol 5mL, scale is diluted and be settled to 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, 500 μ g/L phases
The matrix matching standard solution answered.Matrix matching standard solution answers matching while using.
3. the pre-treatment of sample
Weigh green tea extract tablet or capsule 1.0g (being accurate to 0.01g) to fill in polystyrene centrifuge tube in 10mL tools,
The water of 45 DEG C ± 5 DEG C of 5mL 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 60s.Add 1.0g anhydrous magnesium sulfates, ceramics
Matter stone, 0.3g anhydrous sodium acetates, vortex 30s, mechanical shaking extraction 1min.Centrifuge tube is placed in into centrifuge, temperature is set as 4 DEG C, with
2264r/min is centrifuged 5min, takes 200 μ L filtrates in 2mL sample introduction bottles, add 500 μ L 8mmol/L formic acid aqueous ammoniums and
300 μ L methanol.It is measured with Ultra Performance Liquid Chromatography-level Four bar-orbit ion trap mass spectrum.
136 kinds of different analyzed detections of green tea health food are had, these products are obtained from different retail trade points,
And be analyzed according to the method described above.
4. analysis result
(1) foundation of method
Sample 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-four
The mass spectrographic full scan pattern of level bar electrostatic field orbit ion trap obtains first mass spectrometric figure, and extracting ion flow graph carries out quantitatively, passing through
Variable data non-dependent collection vDIA Mode scans obtain second order mses figure.Two grades of scannings are obtained on the basis of full scan
More targetedly fragmentation fragment data, you can tentatively judge the material belonging kinds, then to the retention time chromatographic peak
Other piece segment informations are analyzed, including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, so as to be inferred to the material and unit
Element composition.The fragment dissociation pathways that the material is inferred are composed in storehouse with Natural antioxidant standard information with the vitamin set up
Other compound dissociation pathways of ownership class are compared, and judge molecular formula and the structural formula of the compound, by analytical standard material
Chromatographic mass spectrometry information carry out the final composition judged with vitamin in quantitative green tea extract health product with the Natural antioxidant.
(2) Method validation
The standard curve of a methods, the range of linearity and correlation coefficient
52 kinds of vitamin are prepared with Natural antioxidant series hybrid standard substance solution, concentration is 0.1 μ g/kg, 0.5 μ
g/kg、1μg/kg、2μg/kg、4μg/kg、8μg/kg、10μg/kg、20μg/kg、40μg/kg、80μg/kg、100μg/kg、200
μg/kg、400μg/kg、500μg/kg.As a result show, in detection method, 52 kinds of vitamin are corresponding at which with the Natural antioxidant
In 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 present invention carries out method to the approach application EU criteria 2002/657/EC and SANCO/12571/2013 for having set up
Learn parameter to investigate：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
2.33 times of corresponding repeatability standard deviation.1.64 times of repeatability standard deviation when CC β add CC α values for the value of CC α.Really
Fixed limit (CC α) and measurement capacity (CC β) are respectively 0.05 μ g/kg-1.91 μ g/kg and 0.08 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, calculate 52 kinds of vitamin and the Natural antioxidant average recovery rate and
Relative standard deviation.The response rate and relative standard deviation result are respectively 85%-113% and 0.3%-6.4%, the side for being set up
Method accuracy and precision are good.Table 2 below lists the side of vitamin and the Natural antioxidant in green tea extract health product
Science of law parameter.
The range of linearity of vitamin and the Natural antioxidant in 2 green tea extract health product of table, determine limit, measurement capacity,
Correlation coefficient, average recovery rate and precision (n=6)
(3) result
The present invention is optimized to detection method and verifies, experimental subject is that 136 kinds of green tea for taking from different markets are carried
Take thing health product.The present invention has carried out Screening analysis to 52 kinds of vitamin and the Natural antioxidant, as a result shows, vitamin, resists
Bad hematic acid is present in a large number, and other vitamin, and such as vitamin A and tretinoin is not detected among out；In the Natural antioxidant,
The content of (-)-epigallo catechin catechu, epigallocatechin gallate (EGCG) and epicatechin is higher than caffeic acid and green
Ortho acid；Quantity that health-care product contains composition and indicate on label similar (thiamine is less than indicating on the label declared
Quantity；Label value of the vitamin B in sample than providing is high)；And contain caffeic acid in all analyzed samples.
The screening method of the present invention can also be applied to the examination of vitamin and the Natural antioxidant in other health product.
Fig. 1 interaction diagrams intuitively reflect impact effect of each factor of QuEChERS pre-treatments to response value (response rate)
Really, the optimal parameter of Pretreatment is：Add 10mL 1% (v/v) acetic acidacetonitrile/water (84:16, v/v) mixed solution, second
Sour sodium usage amount is 0.30g, by the theoretical value of the regression equation predictive compound response rate up to 99.33%.Using above-mentioned optimization
The Pretreatment for going out carries out 6 experiments, and it is 99.28% to measure response rate meansigma methodss, and deviation is less, is shown using the response surface point
The QuEChERS Pretreatments parameter that the optimization of analysis method is obtained is accurately and reliably.Fig. 2 can be seen that oleanolic acid (Oleanic
Acid) 8.37min and 8.39min is respectively with ursolic acid (Ursolic acid) retention time, molecular ion mass-to-charge ratio is
457.36762.Fragmentation fragment is identical, but abundance ratio is different, can be 203.17937 and 297.25735 two by mass-to-charge ratio
The response intensity of fragmentation fragment is relative to be distinguished with abundance ratio and is confirmed.
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 be in national standard in health product mutually outside the Pass
The quantitative limit setting 10 of source property material mass spectrometric determination, chooses vitamin and Natural antioxidant mark in the health product set up
Quasi- 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 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 carry
Response intensity is got more than or equal to 8.3 × 104During chromatographic peak, sample is tentatively judged as positive, carries out next step confirmation work
Make, 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 enters
The extraction and analysis of row feature fracture fragment, carries out Screening analysis to unknown object material, and then reaches a conclusion.Confirmation work master
To be carried out by two aspects of isotope ion peak information and two grades of fragmentation fragment abundance ratios, and solve isomerss chromatograph
The confirmation difficult point for flowing out altogether, by with M+1 features13C, and with M+2 features34S、81Br、37The isotope ion peak of Cl is rich
Degree is than being applied to confirm step.
At present for the research of vitamin in health product and natural anti-oxidation agent high flux rapid screening technology is still blank.
, the pre-treatment complex operation less with the high flux, method for quick that the Natural antioxidant is remained for vitamin in health product,
Material is high with equipment cost, is only limitted to several or a class compound quick detection, remains when quickly analyzing often according to change more
The construction featuress of compound are detected after compound is grouped.In recent years, the application of QuEChERS wide spectrums extracting method and pass through instrument
Combination causes health product exogenous specified risk material Screening analysis technology to develop rapidly.Herein for above-mentioned present situation, with high score
Mass spectrum level Four bar electrostatic field orbit ion trap mass spectrum is distinguished, and combines Chemical Measurement and QuEChERS Pretreatments are optimized and is determined
Amount data analysiss, for the health product closely bound up with people's life, establish exogenous specified risk material combined gas chromatography mass spectrometry high
Flux, rapid screening analysis method.
With health product complex matrices as model, exogenous specified risk material known target material Screening analysis method is established.
With the methodology parameter plan of survey of system-EU criteria 2002/657/EC and SANCO/12571/2013, to using ultra high efficiency
Liquid chromatograph-level Four bar electrostatic field orbit ion trap mass spectrum, the examination of vitamin and the Natural antioxidant in the health product of foundation
Analysis method is examined, and in all storehouses, potential target compound has good linear relationship, phase relation in linear scope
Number is all higher than 0.99, under high, medium and low three pitch-based spheres, and the response rate reaches 85%-113%, repeats experiment 6 times, its relative mark
Quasi- deviation is 0.3%-6.4%.It is determined that limit (CC α) and measurement capacity (CC β) are respectively 0.05 μ g/kg-1.91 μ g/kg and 0.08
μ g/kg-3.18 μ g/kg, better than the limitation requirement that the country such as China, Japan, USA and EU and relevant international organization specify, spirit
Sensitivity 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 (8)
1. in a kind of health product vitamin and the Natural antioxidant Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap
Mass spectrum screening method, it is characterised in that：Comprise the following steps：
1) the measured object sample of health product is extracted and is purified using QuEChERS methods, obtained the test of measured object sample
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, extract in measured object sample vitamin with it is natural
The compound information of antioxidant；
4) by the compound information in vitamin and Natural antioxidant standard spectrum storehouse and the vitamin of measured object sample with it is natural
Anti-oxidant compounds information is compared, and is finally judged and is quantitatively protected by the chromatographic mass spectrometry information of analytical standard material
The composition of vitamin and the Natural antioxidant in strong product.
2. in health product according to claim 1 vitamin and the Natural antioxidant Ultra Performance Liquid Chromatography-level Four bar
Electrostatic field orbit ion trap mass spectrum screening method, it is characterised in that：Described health product are green tea extract health product.
3. in health product according to claim 1 and 2 vitamin and the Natural antioxidant 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：Weigh green tea extract tablet or glue
Capsule adds 45 DEG C ± 5 DEG C of water in centrifuge tube, and being vortexed fully to mix is completely dispersed sample, cools down in being put into circulating water；Again
Add the acetic acidacetonitrile/water mixed solution of volume fraction 1%, vortex mixed；Add anhydrous magnesium sulfate, ceramic homogenizing stone, anhydrous
Sodium acetate, vortex, mechanical shaking extraction；After finally be centrifuged centrifuge tube, take filtrate in sample introduction bottle, add formic acid aqueous ammonium and
Methanol.
4. in health product according to claim 1 vitamin and the Natural antioxidant 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：By Ultra Performance Liquid Chromatography-level Four
The mass spectrographic full scan pattern of bar electrostatic field orbit ion trap obtains first mass spectrometric figure, and extracting ion flow graph is carried out quantitatively, by can
The data non-dependent collection vDIA Mode scans of change obtain second order mses figure.
5. in health product according to claim 4 vitamin and the Natural antioxidant 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 that be inferred to the structure of matter and elementary composition.
6. in health product according to claim 1 vitamin and the Natural antioxidant Ultra Performance Liquid Chromatography-level Four bar
Electrostatic field orbit ion trap mass spectrum screening method, it is characterised in that：Step 4) in vitamin and Natural antioxidant standard spectrum
Storehouse is set up according to following steps：
1) standard solution of vitamin and Natural antioxidant standard substance is prepared respectively；
2) with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph to vitamin and Natural antioxidant mark
The standard solution of quasi- product 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 standard
Spectrum storehouse.
7. in the health product according to claim 1 or 4 vitamin and the Natural antioxidant 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 orbiting ions
The mass spectrometric chromatographic condition of trap 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 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：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：230-250
℃；Capillary temperature：320-330℃.
8. in health product according to claim 1 vitamin and the Natural antioxidant 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 spectrometric Mass Spectrometry Conditions 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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