CN107860858A - A kind of method for high-flux analysis of mycotoxin in plant medicine material - Google Patents
A kind of method for high-flux analysis of mycotoxin in plant medicine material Download PDFInfo
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- G—PHYSICS
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- 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
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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
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Abstract
The invention provides a kind of method for high-flux analysis of the mycotoxin in plant medicine material, including step：The pre-treatment of plant medicine material powder；UHPLC MS/MS are analyzed.The present invention is directed to the matrix feature of plant medicine material, dexterously replaces the aqueous solution using acidic buffer, reduces the concentration of required formic acid, so as to reduce the extraction of acid impurities, effectively reduces interference；Silica filler is reasonably introduced, removing polarity in plant medicine material matrix for specific aim relatively strengthens compound, avoids using conventional filler GCB, effectively improves the mycotoxin rate of recovery；The present invention establishes the UHPLC MS/MS analysis systems and method that 5 class Pseudomonas amount to the qualitative, quantitative of 70 kinds of mycotoxins first, significantly improve plant medicine material sample pre-treatments level of purification and all kinds of mycotoxin rate of recovery, mycotoxin monitoring species has significantly been expanded, therefore has been had broad application prospects and good market potential.
Description
Technical field
The invention belongs to Chinese medicine to analyze detection field, and in particular to a kind of height of the mycotoxin in plant medicine material
Throughput analytical methods.
Background technology
Mycotoxin, it is the toxic metabolic products of fungi, which part mycotoxin toxicity is huge, or even can cause carcinogenic
Etc. strong effect.It is well known that mycotoxin is the important pollutant in food and feed, by the World Health Organization
(World Health Organization, WHO) is classified as the important root of food origin disease；Also, mycotoxin can be by straight
Connect intake, suction and skin contact to enter in human body and livestock body, there is serious chronic toxicity, including liver, renal toxicity, cause
Carcinous, mutagenicity and teratogenesis etc..For example, aflatoxin B1It is set to because of its extremely strong carcinogenicity by the World Health Organization
A kind of carcinogenic substance, serious threat is caused to human health.
Also there is the phenomenon that mycotoxin pollutes in Chinese medicine, harm is definite, is increasingly subject to pay attention to.Chinese medicine category can be divided into
Plant, animal class and mineral substance, wherein plant medicine material kind are most, and usage amount is most extensive.The root of plant, stem, leaf,
Flower, each position of fruit can be used as medicine, and such medicinal material is harsh for the temperature and humidity conditions stored, transported, and potential mouldy microbiological contamination be present
So as to the risk of contaminant toxin.According to the literature, it has been found that the phenomenon for infecting a variety of mycotoxins in Chinese medicine be present.By
It is huge in China's usage amount in Chinese medicine, and the prescriptions of traditional Chinese medicine of the overwhelming majority is related to plant medicine material, while Part of Chinese Medicinal quilt
The public thinks with health role, harm caused by service life is some months even several years, therefore mycotoxin pollutes
It can not be ignored；Especially some fungi toxin is not easy to be metabolized with savings property, and the Chinese medicine polluted by mycotoxin is eaten for a long time
Easily cause serious chronic toxicity, therefore government and the public are to the pollution pay attention to day by day of mycotoxin in Chinese medicine.
Mycotoxin limit standard is less in plant medicine material, is badly in need of working out, detection demand sharply increases.With to true
The attention of verticillium toxin and going deep into for research, the mycotoxin species being currently known are up to kind more than 400.The toxin paid close attention to both at home and abroad
Species is not limited to original well known more than ten and planted already, the toxicity research knot of the increasing various mycotoxins of reported literature
Fruit, increasing mycotoxin are found and determine that its is harmful.However, although the whole world has had more than 100 countries
The limit standard about mycotoxin has been formulated, but has been only limitted to aflatoxin etc. more than 10 and plants mycotoxin, and has concentrated on food
Field of fodder, then there was only aflatoxin G in Chinese medicine1、G2、B1、B2Four kinds of limitation regulations.However, with to Chinese medicine safety
Property pay attention to day by day, increasingly urgent, substantial amounts of basis detection data are worked out to the limit standard of various mycotoxins in Chinese medicine
It is badly in need of collecting.Therefore, fast and efficiently mycotoxin detection method is badly in need of establishing a kind of high throughput analysis side of mycotoxin
Method.
Variety classes mycotoxin chemical constitution is different, and physicochemical property difference is larger, therefore, establishes a variety of mycotoxins
High throughput method needs rational chromatographic detection method and effective sample-pretreating method.Mycotoxin is mainly produced by 5 major classes
Malicious fungal metabolite produces, and different mycotoxin chemical constitutions is different, including coumarin derivatives, long-chain di esters, class are female
Steroids, indoles alkaloid derivative, macrolides etc., difference is big, and physicochemical property is different, and this is established high flux
Mycotoxin detection method bring great difficulty.Ultra performance liquid chromatography and multi-stage mses GC-MS (UHPLC-MS/
MS) mycotoxin of different nature can be detected simultaneously, but the species for detecting toxin simultaneously is more, the chromatogram of development system
The difficulty of detection method is bigger.Particularly, mycotoxin generally exists in Chinese medicine in the form of trace even ultra trace, from
And the sensitivity and pre-treatment to detection method propose harsher requirement, it is seen then that the key effectively to solve the above problems is
Component to be measured how is effectively enriched with, while removes chaff interference as much as possible using the key as pre-treatment.
In addition, Chinese medicine matrix is complicated, mycotoxin detects in food sample pre-treatments and chromatogram detection can not be applied mechanically
Method, especially more toxin high flux detections belong to forward position research, more need further to innovate.Mycotoxin in Chinese medicine field at present
Detection be still in the ground zero stage, and focus primarily upon the research of a few well known mycotoxin.Fungi poison in food
The research of element is more early, and the principal component of food is moisture, starch, protein and grease, it is seen that food substrate is relatively easy；With this
Difference, Chinese medicine matrix are mainly dry, the various secondary metabolites rich in plant, so fungi in food can not be applied mechanically
The pre-treatment of Mycotoxin identification and chromatographic detection method, it is necessary to develop applicable new method.
In the prior art, the tradition extraction and purification method for being directed to mycotoxin in Chinese medicine both at home and abroad mainly have immune parent
With post method, solid phase extraction techniques method etc..But the immune affinity column of commercialization is also suitable only for most more than 10 kind mycotoxins
Sample pre-treatments, and prices are rather stiff for the immune affinity column of All-in-One；Solid phase extraction techniques are cumbersome, time-consuming and to technology
Personnel requirement is higher.By contrast, the QuEChERS sample-pretreating methods occurred in the world in recent years by its cost it is cheap,
Efficiently, simple and green safe advantage is increasingly becoming a kind of effective technological means.
The sample pre-treatments that the earliest development and application of QuEChERS methods detect in vegetables and pesticide residues in fruits.It is existing
QuEChERS methods are mainly made up of extraction and purification two benches；Wherein, the extraction stage mainly includes, and first adopts and is soaked in water, then
Extracted with acetonitrile solvent, cleansing phase then carries out adsorption-edulcoration using different fillers, and the filler mainly used has N- propyl group second two
Amine (primary secondary amine, PSA), C18With Graphon (graphite carbon black, GCB).
Also there are the sample pre-treatments that a small amount of document report carries out mycotoxin detection using QuEChERS methods at present, such as
“ZHANG SS,JI S,LU JW,et al.Multi-mycotoxins analysis in Pheretima using
ultra-high-performance liquid chromatography tandem mass spectrometry based
on a modified QuEChERS method.Journal of Chromatography B,2016,1035:31-41 ", this
Document report using QuEChERS pre-treating methods to 22 kinds of mycotoxins in Chinese traditional medicine sleeper determine simultaneously.The document
Method employs traditional PSA, C18With tri- kinds of purification fillers of GCB, used Extraction solvent is to traditional Extraction solvent acetonitrile
Increase acidity so that the concentration of formic acid has reached 15%, and as a result the rate of recovery of most of mycotoxin basically reaches international logical
Required with the rate of recovery：80%~120%, but the rate of recovery of Fumonisins mycotoxin is not ideal enough, such as fumonisins B1Return
Yield is only 73%.
However, document report method is not suitable for the detection of more high-throughout a variety of mycotoxins in plant medicine material
And analysis.Because the research object earthworm of this document is animal tcm material, and it only have studied Eurotium, Fusarium two
Class amounts to 22 kinds of mycotoxins；The method is applied to plant medicine material by inventor, while the mycotoxin kind detected increases
The mycotoxin of Claviceps, Penicillium and rod method Pseudomonas is added, 5 big classifications amount to 70 kinds of mycotoxins, resulting
Method validation result is unsatisfactory, and matrix interference is larger, and the rate of recovery of most of mycotoxin is all unable to reach international time
Yield requirement.In addition, the formic acid concn in the Extraction solvent of document report method is higher, easily extract more acid miscellaneous
Matter, increase so as to disturb so that the baseline rise of plurality of target analyte detection, influence test limit so that sensitivity can not meet to examine
The demand of survey.
Therefore, a kind of brand-new method for high-flux analysis for being directed to mycotoxin in plant medicine material is developed, simultaneously
Ensure the satisfied mycotoxin rate of recovery, and realize accurate quantification and qualification, become current this area research staff
One of research emphasis.
The content of the invention
In order to overcome technological deficiency present in prior art, the present invention is based on traditional QuEChERS methods, pin
To the matrix feature of plant medicine material, the aqueous solution is dexterously replaced using acidic buffer, while changes Extraction solvent system,
The concentration of formic acid, so as to reduce the extraction of acid impurities, effectively reduces interference needed for reducing；The present invention also reasonably draws
Silica filler is entered, removing polarity in plant medicine material matrix for specific aim relatively strengthens compound, is such as difficult to the saponin(e removed
Class, flavonoids and alkaloids impurity, while for avoiding using conventional filler GCB, effectively improve mycotoxin recovery
Rate, clean-up effect is improved, and be added significantly to detectable mycotoxin species in plant medicine material.The present invention is simultaneously
The analyzing detecting method for 70 kinds of mycotoxins in plant medicine material is established with reference to UHPLC-MS/MS technologies, there is height
The beneficial characteristics such as sensitivity, quick detection, high flux.
Specifically, the invention provides a kind of method for high-flux analysis of the mycotoxin in plant medicine material, including
Following steps：
S1：The pre-treatment of plant medicine material powder；
S2：UHPLC-MS/MS is analyzed；
Wherein, the pre-treatment described in S1 includes：
It is accurately weighed and take the plant medicine material powder, it is placed in centrifuge tube, precision adds13C- deoxynivalenols
Bacterium enol inner mark solution and13C- zearalenone inner mark solutions, are subsequently added into acetate buffer, are vortexed；It is accurate again to add
The acetonitrile solution of 5% formic acid, is vortexed again for, and is subsequently placed in Ultrasound Instrument ultrasonic；It is subsequently added into anhydrous magnesium sulfate and sodium chloride
Mixed-powder, shake immediately scattered；It is subsequently placed on high-speed oscillator and acutely shakes, centrifuged after ice bath, pipettes supernatant and silicon extremely is housed
Glue, PSA, C18 and MgSO4Solid phase extraction pipe in, after vortex, be placed on high-speed oscillator and acutely shake, centrifuge again；
Accurate Aspirate supernatant, nitrogen blow, and are redissolved with methanol aqueous solution, after vortex, with filtering with microporous membrane, take subsequent filtrate, obtain sample introduction
Liquid.
What deserves to be explained is the method for high-flux analysis of above-mentioned mycotoxin has abandoned GCB fillers of the prior art, into
Work(introduces silica filler so that it is complete with C18, N- propyl group ethylenediamine (primary secondary amine, PSA) composition
Combination nova filler, the purification pre-treatment for sample.The silica filler can adsorb the stronger compound of polarity, specific aim absorption
It is difficult to the impurity such as the saponins that removes in plant medicine material, clean-up effect not only greatly improved, and effectively evaded stone
Some fungi toxin recovery caused by the use meeting of inkization carbon black (graphite carbon black, GCB) filler significantly drops
The problem of low.For a large amount of pigments present in plant medicine material and amount of grease class low pole impurity, above-mentioned mycotoxin
Method for high-flux analysis pointedly used PSA and C18, and filling mixture ratio is optimized.In addition, the present invention is rationally
Using LC-MS technology (UHPLC-MS/MS) establish first Eurotium, Fusarium, Claviceps, Penicillium and
The Mass Spectrometry detection method of Alternaria 5 class Pseudomonas of element totally 70 kinds of mycotoxins, covers current mycotoxin research field substantially
The mycotoxin classification of Literature report, 80% of mycotoxin sum in current European Union's food contaminant tracking list is accounted for,
Which includes the mycotoxin species of newest concern in recent years.
Preferably, in above-mentioned method for high-flux analysis, the water used in S1 is the anaerobic water now matched somebody with somebody, by by nitrogen
It is passed through in deionized water more than 15 minutes and is made.
Preferably, it is described in above-mentioned method for high-flux analysis13The concentration of C- deoxynivalenol inner mark solutions
It is described for 5mg/L13The concentration of C- zearalenone inner mark solutions is 5mg/L.
Preferably, in above-mentioned method for high-flux analysis, the pH of the acetate buffer is 3.0.
Preferably, in above-mentioned method for high-flux analysis, the mass ratio of the anhydrous magnesium sulfate and sodium chloride is 4:1.
Preferably, in above-mentioned method for high-flux analysis, the rotating speed of the centrifugation is 5000~12000r/min.
Preferably, in above-mentioned method for high-flux analysis, the UHPLC-MS/MS analyses described in S2 are included the sample introduction
Step in liquid sample introduction to ultra performance liquid chromatography and multi-stage mses combined system；Wherein, the bar of the ultra performance liquid chromatography
Part includes：
Agilent Poroshell 120EC-C18 chromatographic columns, 150mm × 2.1mm I.D, 2.7 μm；
Under positive ion mode, using the aqueous solution containing 0.4% formic acid and 2mM ammonium formates as mobile phase A, to contain 0.4%
The methanol solution of formic acid and 2mM ammonium formates is Mobile phase B, flow velocity 0.45ml/min, and carries out gradient elution by following procedure：
0min, 80%A, 20%B；2min, 80%A, 20%B；6min, 50%A, 50%B；11min, 45%A, 55%B；15min,
100%B；18min, 100%B；20min, 80%A, 20%B；25min, 80%A, 20%B；
Under negative ion mode, using water as mobile phase A, acetonitrile is Mobile phase B, flow velocity 0.45ml/min, and presses following journey
Sequence carries out gradient elution：0min, 90%A, 10%B；2min, 90%A, 10%B；8min, 50%A, 50%B；13min, 40%
A, 60%B；15min, 100%B；17min, 100%B；18min, 90%A, 10%B；20min, 90%A, 10%B.
It is further preferred that in above-mentioned method for high-flux analysis, the condition of the multi-stage mses includes：Positive ion mode
Under：Ion gun：Electric spray ion source；Scan mode：Positive ion mode；Detection mode：Multiple-reaction monitoring pattern；Electron spray electricity
Pressure：5500V；Atomization gas pressure：50.0psi；Assist gas pressure power：50.0psi；Gas curtain atmospheric pressure：30.0psi；Collide atmospheric pressure：
7.0psi；Ion source temperature：450℃；Collision cell entrance potential：10V；Collision cell exit potential：14V；
Under negative ion mode：Ion gun：Electric spray ion source；Scan mode：Negative ion mode；Detection mode：More reactions
Monitoring pattern；Electron spray voltage：-4500V；Atomization gas pressure：50.0psi；Assist gas pressure power：50.0psi；Gas curtain atmospheric pressure：
30.0psi；Collide atmospheric pressure：7.0psi；Ion source temperature：400℃；Collision cell entrance potential：-10V；Collision cell outlet electricity
Pressure：-12V.
Compared with prior art, technical scheme provided by the present invention has the advantages that：
(1) the matrix feature of plant medicine material is directed to, the aqueous solution is dexterously replaced using acidic buffer, so as to compare
There is following technical advantage in aqueous phase system of the prior art：
1. the concentration of formic acid in Extraction solvent acetonitrile can be significantly reduced, so as to the extraction to reducing acid impurities, for example,
Found when being applied to other plant medicine materials using the formic acid of 20% concentration described in document, the interference by target analytes
Summit showed increased；2. soaking solvent as test sample using acidic buffer, stabilization in Extraction solvent acetonitrile can be maintained
Formic acid concn, with more universality；It is different additionally, due to material base of plant medicine material itself, to the pH value of Extraction solvent
It is required that it is also different, conventional method pre-treatment such as is used, that is, adopts the test sample that is soaked in water, then required Extraction solvent may require that addition
Different acidity, such as radix glycyrrhizae with the acetonitrile solution optimum of 15% formic acid, and for PERICARPIUM TRICHOSANTHIS and pseudo-ginseng, the first of 10% concentration
Acid is enough, and lotus seeds then show optimal effectiveness in the formic acid of 5% concentration；The present invention is used with certain buffer capacity
Acidic buffer soaks sample, is able to maintain that the pH value of Extraction solvent so that formic acid concn maintains 5% in Extraction solvent；③
The acidic buffer that the present invention uses can effectively improve the rate of recovery of fumonisins；Fumonisins is the very high one kind weight of attention rate
The mycotoxin wanted, its acidity to Extraction solvent is extremely sensitive, under conditions of pH is sufficiently low, can just obtain gratifying
The rate of recovery；The fumonisins rate of recovery of pertinent literature report is not high, and the present invention can obtain preferable under relatively low formic acid concn
The fumonisins rate of recovery；
(2) silica filler is rationally used, to avoid using conventional filler GCB；With conventional purge filler GCB, PSA and C18
Compare, silica filler has stronger adsorption effect for the larger impurity of polarity be present in plant medicine material, so as to significantly
Improve the rate of recovery of target toxin；
(3) Isotopic Internal Standard of mycotoxin to be measured is rationally used13C- deoxynivalenols and13C- corns are red
Mould ketenes, the difference responded to correcting sample pre-treatment operation and mass spectrometer, has been effectively ensured the accuracy of method calculating；
(4) the UHPLC-MS/MS analysis bodies of qualitative, quantitative while 5 class Pseudomonas amount to 70 kinds of mycotoxins are established first
System and method, the most high flux inspection of mycotoxin kind is provided for detection and the analysis of the mycotoxin in current Chinese medicine
Survey technology.
In summary, the method for high-flux analysis of the mycotoxin in plant medicine material of the present invention significantly improves
Plant medicine material sample pre-treatments level of purification and all kinds of mycotoxin rate of recovery, mycotoxin monitoring kind is significantly expanded
Class, therefore have broad application prospects and good market potential.
Brief description of the drawings
Fig. 1 is that some fungi toxin after literature method (a) and the inventive method (b) processing radix glycyrrhizae sample is respectively adopted
15-ADON qualitative ion pair chromatograms；
Fig. 2 is that some fungi toxin after literature method (c) and the inventive method (d) processing radix glycyrrhizae sample is respectively adopted
15-ADON quota ion pair chromatograms；
Fig. 3 is that some fungi toxin after literature method (e) and the inventive method (f) processing radix glycyrrhizae sample is respectively adopted
Aflatoxin B2Qualitative ion pair chromatogram；
Fig. 4 is that some fungi toxin after literature method (g) and the inventive method (h) processing radix glycyrrhizae sample is respectively adopted
Aflatoxin B2Quota ion pair chromatogram；
Fig. 5 is to purify the radix glycyrrhizae matrix total ion current figures after fillers purify before purifying and using different, wherein：Upper figure
For the total ion current figure under positive ion mode, figure below is the total ion current figure under negative ion mode.
Embodiment
With reference to embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiment party
Formula.
In a preferred embodiment, a kind of method for high-flux analysis of the mycotoxin in plant medicine material include with
Lower step：
S1：The pre-treatment of plant medicine material powder；
S2：UHPLC-MS/MS is analyzed；
Wherein, the pre-treatment described in S1 includes：
It is accurately weighed and take the plant medicine material powder 2g (cross No. three sieve), be placed in 50ml centrifuge tubes, precision plus
Enter 50 μ L 5mg/L's13C- deoxynivalenols inner mark solution and 50 μ L 5mg/L's13C- zearalenone internal standards
Solution, is subsequently added into acetate buffer (pH=3.0) 15ml, and vortex makes sample fully infiltrate；Precision adds 5% formic acid again
Acetonitrile solution 10ml, it is vortexed again for making mixing, is subsequently placed in Ultrasound Instrument ultrasonic 5 minutes (53kHz)；It is subsequently added into anhydrous slufuric acid
The mixed-powder of magnesium and sodium chloride (mass ratio 4:1) 5g, shake immediately scattered；Acutely concussion 5 minutes are subsequently placed on high-speed oscillator,
(5000r/min) is centrifuged after ice bath 10min 5 minutes, pipette supernatant 6ml to equipped with 150mg silica gel, 300mg PSA, 300mg
C18 and 900mg MgSO4Solid phase extraction pipe in, be vortexed fully mix after, be placed on high-speed oscillator acutely concussion 5 points
Clock, then centrifuge (12000r/min) 10 minutes；Accurate Aspirate supernatant 2.0ml, nitrogen are blown to below 0.5ml, then use methanol-water
Solution (methanol：Water=20：80, v/v) redissolve to 1.0ml, be vortexed after mixing, filtered, taken with miillpore filter (0.22 μm of aperture)
Subsequent filtrate, produce into sample liquid.
In a preferred embodiment, the water used in S1 is the anaerobic water now matched somebody with somebody, by the way that nitrogen is passed through into deionization
In water more than 15 minutes and be made.
In a preferred embodiment, the UHPLC-MS/MS analyses described in S2 include by it is described enter sample liquid sample introduction to super
High performance liquid chromatography and the step in multi-stage mses combined system；Wherein, the condition of the ultra performance liquid chromatography includes：
Agilent Poroshell 120EC-C18 chromatographic columns, 150mm × 2.1mm I.D, 2.7 μm；Positive ion mode
Under, using the aqueous solution containing 0.4% formic acid and 2mM ammonium formates as mobile phase A, with the first containing 0.4% formic acid and 2mM ammonium formates
Alcoholic solution is Mobile phase B, flow velocity 0.45ml/min, and carries out gradient elution by table 1；Under negative ion mode, using water as mobile phase
A, acetonitrile are Mobile phase B, flow velocity 0.45ml/min, and carry out gradient elution by table 2.
Condition of gradient elution under the positive ion mode of table 1
Condition of gradient elution under the negative ion mode of table 2
Wherein, the condition of the multi-stage mses includes：
(internal standard under positive ion mode：13C- deoxynivalenols)：Ion gun：Electric spray ion source；Scanning side
Formula：Positive ion mode；Detection mode：Multiple-reaction monitoring pattern；Electron spray voltage：5500V；Atomization gas pressure：50.0psi；It is auxiliary
Help atmospheric pressure：50.0psi；Gas curtain atmospheric pressure：30.0psi；Collide atmospheric pressure：7.0psi；Ion source temperature：450℃；Collision cell
Entrance potential：10V；Collision cell exit potential：14V；
(internal standard under negative ion mode：13C- zearalenones)：Ion gun：Electric spray ion source；Scan mode：Bear from
Subpattern；Detection mode：Multiple-reaction monitoring pattern；Electron spray voltage：-4500V；Atomization gas pressure：50.0psi；Assist gas pressure
Power：50.0psi；Gas curtain atmospheric pressure：30.0psi；Collide atmospheric pressure：7.0psi；Ion source temperature：400℃；Collide chamber inlet electricity
Pressure：-10V；Collision cell exit potential：-12V.
Also, prepare the standard liquid (100~500mg/L) of the debita spissitudo of 70 kinds of mycotoxins respectively using acetonitrile,
And pin pump is used under manual tuning patterns, with current constant mode sample introduction.In Analyst software 1.6.2 (AB
SCIEX the mass-to-charge ratio of parent ion) is determined on software using Q1Scan, is accurate to one decimal place；Appropriate concentration is controlled, to the greatest extent
The mass spectra peak response of object may be made between 1e6~3e6cps；With 100~M+50 of scanning range (relative molecular mass+
50) and sweep speed 200Da/s is scanned, and under cation scan pattern, is found and is responded best [M+H]+Or [M+NH4]+
Peak is as parent ion；Under anion scan pattern, find and respond best [M-H]-Or [M+HCOO]-Peak.Then in Product
Under Ion Scan patterns, selected target parent ion sets CE initial values as 5eV, and CE, parent ion are adjusted manually by step-length of 5eV
Intensity be that base peak intensity 1/3 to 1/4 is advisable in collection of illustrative plates, 2~3 good and stable daughter ions of Response to selection, and judgement is split
The possibility of solution.According to above select mother, daughter ion, set up MRM ion pairs, every a pair of analysis time is arranged to 100ms；
With the mode automatically optimized collision energies of Ramp (Collision Energy, CE) and remove cluster voltage (Declustering
Potential, DP), the 70 kinds of mycotoxins and the mass spectrometry parameters of 2 kinds of inner mark solutions finally given are as shown in table 3 below：
The mycotoxin mass spectrometry parameters table of table 3
In addition, as shown in figure 1, compared to the total ion current figure for employing matrix after GCB is purified, it is net to employ silica gel (Si)
After change in the total ion current figure of matrix, either under positive ion mode or negative ion mode, substantially reduced at the appearance of front end
Ambient interferences, i.e., impurity clean-up effect larger to polarity significantly improve.Because GCB primary attachments pigment and small
Particle, surface hexagonal structure cause its molecule for planar molecule or containing plane aromatic rings that there is strong absorption to make
With, and due to its imporosity, the absorption to sample does not require that it diffuses to hole area, so GCB adsorption process is very
Rapidly, it adsorbs total capacity and is also twice than silica gel and is had a surplus, therefore GCB easily causes to adsorb non-selectivity, and ultimately causes mesh
The rate of recovery of mark analyte is greatly reduced.Unlike this, present invention introduces silica gel, be present substantial amounts of silanol base in its surface, hold
Hydrogen bond easily is formed with polar substances, so as to optionally adsorbing contaminant, does not interfere with the rate of recovery of mycotoxin.
Embodiment 1
Inventor takes certain factory's radix glycyrrhizae sample according to method for high-flux analysis provided by the present invention to 70 kinds of fungies in radix glycyrrhizae
Toxin carries out extraction separation and detected with analysis, the range of linearity and test limit (LOD) such as table 4 below of resulting 70 kinds of mycotoxins
Shown, the rate of recovery of 70 kinds of mycotoxins is as shown in table 5 below.
The range of linearity and test limit of the mycotoxin of table 4
The rate of recovery of the mycotoxin of table 5
Also, inventor is also by method for high-flux analysis in accordance with the present invention to true obtained by factory's radix glycyrrhizae sample analysis
The verticillium toxin rate of recovery and document " ZHANG SS, JI S, LU JW, et al.Multi-mycotoxins analysis in
Pheretima using ultra-high-performance liquid chromatography tandem mass
spectrometry based on a modified QuEChERS method.Journal of Chromatography B,
2016,1035:Result under method item described in 31-41 " compares, as a result as shown in table 6 below：
The mycotoxin rate of recovery of table 6 compares
It can be seen that the analysis method that this embodiment is implemented expands detectable mycotoxin species, by original 22 kinds
70 kinds are increased to, with addition of Penicillium, Claviceps and the other mycotoxin of the major class of Alternaria 3.Particularly,
Ochratoxin A、Fumonisin B1、Fumonisin B2With Fumonisin B3The rate of recovery of this 4 kinds of mycotoxins has bright
It is aobvious to improve；Former literature method is respectively 88%, 73%, 84% to this rate of recovery of 4 kinds of mycotoxins under three mark-on levels,
80%, and in this embodiment the corresponding rate of recovery be 93%, 85%, 90%, 93%, and the RSD values between parallel test from
6.9% is improved to 4.1%.
Also, understand that the application of buffer solution system reduces the concentration of formic acid in acetonitrile so that some fungi with reference to Fig. 1
Toxin response increase, reduces interference, so as to reduce miscellaneous peak (by taking 15-Acetyldeoxynivalenol as an example), baseline is more
Steadily；Interference Peaks are few, and improve the response of some fungi toxin (with Aflatoxin B2Exemplified by).
At the same time, the rate of recovery of detectable mycotoxin of the inventor also to increasing species newly compares, that is, distinguishes
It can detect using 31 kinds of the analysis method described in above-mentioned document and method for high-flux analysis of the present invention detection are newly-increased
Mycotoxin, and compare its rate of recovery, it is as a result as shown in table 7 below：
The mycotoxin rate of recovery that table 7 increases species newly compares
It can be seen that international standard is not complyed with using the rate of recovery of most of mycotoxin obtained by document analysis of report method
(80%~120%), and the rate of recovery can be then significantly improved according to the analysis method of the present invention that the present embodiment is carried out, completely
Meet international standard.
In addition, being understood with reference to Fig. 2, silica filler (Si) of the present invention, can optionally adsorb in plant often
The polarity secondary metabolite seen, while the suction-operated of GCB contratoxin is avoided, finally improve the rate of recovery；Specifically, tie
Front and rear sample (being purified respectively with PSA, GCB and Si) will be purified in positive ion mode (m/z respectively by closing Q-TOF high resolution mass spectrums
Sweeping entirely under full scan patterns 100-1100) and under negative ion mode (m/z100-1100) is carried out, as shown in Fig. 2 silica gel
Under the total ion current figure either positive ion mode or negative ion mode of matrix after filler purification, PSA and GCB front ends are compared
The ambient interferences of appearance are minimum, have best clean-up effect.
Embodiment 2
Inventor takes the oldenlandia diffusa sample of certain factory according to analysis method provided by the present invention in oldenlandia diffusa
70 kinds of mycotoxins carry out extraction separation and detected with analysis, and the rate of recovery of resulting 70 kinds of mycotoxins is as shown in table 8 below, can
See that 70 kinds of mycotoxins can obtain the gratifying rate of recovery in oldenlandia diffusa.
The rate of recovery of the mycotoxin of table 8
The specific embodiment of the present invention is described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (8)
1. the method for high-flux analysis of the mycotoxin in a kind of plant medicine material, it is characterised in that comprise the following steps：
S1：The pre-treatment of plant medicine material powder；
S2：UHPLC-MS/MS is analyzed；
Wherein, the pre-treatment described in S1 includes：
It is accurately weighed and take the plant medicine material powder, it is placed in centrifuge tube, precision adds13C- deoxynivalenol bacterium alkene
Alcohol inner mark solution and13C- zearalenone inner mark solutions, are subsequently added into acetate buffer, are vortexed；It is accurate again to add 5% first
The acetonitrile solution of acid, is vortexed again for, and is subsequently placed in Ultrasound Instrument ultrasonic；It is subsequently added into the mixed powder of anhydrous magnesium sulfate and sodium chloride
End, shake immediately scattered；Be subsequently placed on high-speed oscillator and acutely shake, centrifuged after ice bath, pipette supernatant to equipped with silica gel, PSA,
C18 and MgSO4Solid phase extraction pipe in, after vortex, be placed on high-speed oscillator and acutely shake, centrifuge again；Precision is inhaled
Supernatant is taken, nitrogen blows, and is redissolved with methanol aqueous solution, after vortex, with filtering with microporous membrane, takes subsequent filtrate, must enter sample liquid.
2. method for high-flux analysis according to claim 1, it is characterised in that the water used in S1 is the anaerobic now matched somebody with somebody
Water, by the way that nitrogen is passed through in deionized water into more than 15 minutes to be made.
3. method for high-flux analysis according to claim 1, it is characterised in that described13C- deoxynivalenols
The concentration of inner mark solution is 5mg/L, described13The concentration of C- zearalenone inner mark solutions is 5mg/L.
4. method for high-flux analysis according to claim 1, it is characterised in that the pH of the acetate buffer is 3.0.
5. method for high-flux analysis according to claim 1, it is characterised in that the matter of the anhydrous magnesium sulfate and sodium chloride
Amount is than being 4:1.
6. method for high-flux analysis according to claim 1, it is characterised in that the rotating speed of the centrifugation is 5000~
12000r/min。
7. method for high-flux analysis according to claim 1, it is characterised in that the UHPLC-MS/MS analyses described in S2
Including by it is described enter sample liquid sample introduction to ultra performance liquid chromatography and multi-stage mses combined system in step；Wherein, the superelevation
The condition of effect liquid phase chromatogram includes：
Agilent Poroshell 120EC-C18 chromatographic columns, 150mm × 2.1mm I.D, 2.7 μm；
Under positive ion mode, using the aqueous solution containing 0.4% formic acid and 2mM ammonium formates as mobile phase A, to contain 0.4% formic acid
Methanol solution with 2mM ammonium formates is Mobile phase B, flow velocity 0.45ml/min, and carries out gradient elution by following procedure：0min,
80%A, 20%B；2min, 80%A, 20%B；6min, 50%A, 50%B；11min, 45%A, 55%B；15min, 100%B；
18min, 100%B；20min, 80%A, 20%B；25min, 80%A, 20%B；
Under negative ion mode, using water as mobile phase A, acetonitrile is Mobile phase B, flow velocity 0.45ml/min, and is entered by following procedure
Row gradient elution：0min, 90%A, 10%B；2min, 90%A, 10%B；8min, 50%A, 50%B；13min, 40%A,
60%B；15min, 100%B；17min, 100%B；18min, 90%A, 10%B；20min, 90%A, 10%B.
8. method for high-flux analysis according to claim 7, it is characterised in that the condition of the multi-stage mses includes：
Under positive ion mode：Ion gun：Electric spray ion source；Scan mode：Positive ion mode；Detection mode：Multiple-reaction monitoring
Pattern；Electron spray voltage：5500V；Atomization gas pressure：50.0psi；Assist gas pressure power：50.0psi；Gas curtain atmospheric pressure：
30.0psi；Collide atmospheric pressure：7.0psi；Ion source temperature：450℃；Collision cell entrance potential：10V；Collision cell exit potential：
14V；
Under negative ion mode：Ion gun：Electric spray ion source；Scan mode：Negative ion mode；Detection mode：Multiple-reaction monitoring
Pattern；Electron spray voltage：-4500V；Atomization gas pressure：50.0psi；Assist gas pressure power：50.0psi；Gas curtain atmospheric pressure：
30.0psi；Collide atmospheric pressure：7.0psi；Ion source temperature：400℃；Collision cell entrance potential：-10V；Collision cell outlet electricity
Pressure：-12V.
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