CN108226328A - The collaborative detection method of beauvericin and enniatin in grain and its product - Google Patents
The collaborative detection method of beauvericin and enniatin in grain and its product Download PDFInfo
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- CN108226328A CN108226328A CN201711408684.9A CN201711408684A CN108226328A CN 108226328 A CN108226328 A CN 108226328A CN 201711408684 A CN201711408684 A CN 201711408684A CN 108226328 A CN108226328 A CN 108226328A
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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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- 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 collaborative detection method of beauvericin and enniatin in a kind of grain and its product, this method include the following steps：S1 will be dissolved in organic solution mesoscale eddies, oscillation after grain and cereal product smashing processing and stand；S2 takes the supernatant after standing to be diluted with ultra-pure water；Supernatant solid phase extraction concentration after dilution is purified, and collects eluent by S3；Eluent is carried out high performance liquid chromatography separation, mobile phase A selected as ammonium acetate solution, Mobile phase B selected as acetonitrile solvent by S4.Gradient elution, the gradient elution program are：At the 0th~2 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；At the 2nd~3 minute, the volume ratio 40 of mobile phase A and Mobile phase B:60；At the 3rd~19 minute, the volume ratio 30 of mobile phase A and Mobile phase B:70；At the 19th~21 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；S5, to carrying out Mass Spectrometer Method into sample liquid after gradient elution.
Description
Technical field
The invention belongs to Fungi Mycotoxin identification fields, and in particular to beauvericin and grace in a kind of grain and its product
The collaborative detection method of fusanin.
Background technology
Beauvericin (beauvericin, BEA) and enniatin (enniatins, ENNs) be by certain sickle-like bacteria such as
Proliferation sickle-like bacteria (Fusarium proliferatum), Fusarium oxysporum (F.oxysporum) and fusarium avenaceum
(F.avenaceum) etc. after infecting the cereal such as wheat, barley, rye and oat, one kind for being generated under moist and cryogenic conditions
Mycotoxin.According to its chemical constitution, such compound is known as six ester peptides mycotoxins.It is reported that the toxoid has one
Fixed genotoxicity and cytotoxicity can induce chromosome aberration, Sister chromatid exohange and micronucleus formation etc..It meanwhile should
Toxoid still ionizes carrier inhibitor, enzyme inhibitor and oxidative stress derivant.At present, Italy, Spain, bar
The toxoid is detected in the Grain and its products of states such as west, Japan and Iran.Since the toxoid is dirty to grain and its product
The seriousness and generality of dye, in recent years by the common concern of domestic and international scientific circles and administrative department.However China is at present still
Without the report of such toxins checking method and pollution situation in grain and its product.China is grain-production and consumes big country, grain
Food safety is concerning national health and social development.Therefore, it is beauvericin and grace Fusariumsp in monitoring China's grain and its product
The pollution situation of element, there is an urgent need for establish one kind can fast and accurately measure beauvericin and grace Fusariumsp in grain and its product simultaneously
The analysis method of cellulose content.
Invention content
Based on this, it is necessary to provide the cooperation detection side of beauvericin and enniatin in a kind of grain and its product
Method.
The collaborative detection method of beauvericin and enniatin in a kind of grain and its product, this method include following step
Suddenly：
S1 will be dissolved in organic solution mesoscale eddies, oscillation after grain and cereal product smashing processing and stand；
S2 takes the supernatant after standing to be diluted with ultra-pure water；
Supernatant solid phase extraction concentration after dilution is purified, and collects eluent by S3；
Eluent is carried out high performance liquid chromatography separation, mobile phase A selected as ammonium acetate solution, Mobile phase B selection by S4
For acetonitrile solvent.Gradient elution, the gradient elution program are：At the 0th~2 minute, the volume ratio of mobile phase A and Mobile phase B
100:0；At the 2nd~3 minute, the volume ratio 40 of mobile phase A and Mobile phase B:60；At the 3rd~19 minute, mobile phase A was with flowing
The volume ratio 30 of phase B:70；At the 19th~21 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；
S5, to carrying out Mass Spectrometer Method into sample liquid after gradient elution.
A concentration of 1mmol/L~10mmol/L of the ammonium acetate solution in one of the embodiments,.
A concentration of 2mmol/L of the ammonium acetate solution in one of the embodiments,.
Organic solvent described in the step S1 is the first acetonitrile solution in one of the embodiments,.
The acetonitrile volume fraction in first acetonitrile solution is 85% in one of the embodiments,.
The enniatin includes enniatine A, enniatine A in one of the embodiments,1, grace fusarium
Rhzomorph B and enniatine B1At least one of.
The measuring method of mass spectrum is as follows in one of the embodiments,：
Ion source is electric spray ion source, and scan pattern is scanned for cation, and it is 500~600 DEG C to remove solvent temperature, ion
Change voltage is 5000~6000v, and residence time is 100~300ms.
The measuring method of mass spectrum is as follows in one of the embodiments,：
Ion source is electric spray ion source (ESI sources), and scan pattern is scanned for cation, and it is 550 DEG C to remove solvent temperature, from
Sonization voltage is 5500v, and gas curtain atmospheric pressure is 30psi, and collision gas is medium flow rate, and atomization gas pressure is 80psi, and auxiliary heats
Atmospheric pressure is 80psi, and residence time 200ms, detection mode is multiple-reaction monitoring.
The step of solid phase extraction concentration described in the step S3 purifies in one of the embodiments, includes：
S30 activates the solid-phase extraction column used in Solid Phase Extraction；
S31, by the solid-phase extraction column after the supernatant overactivation after being diluted in step S2；
S32 elutes the solid-phase extraction column successively with the second acetonitrile solution of various concentration；And
S33 elutes the solid-phase extraction column with third acetonitrile solution, it is spare to collect the eluent.
The solid-phase extraction column is octadecylsilane chemically bonded silica in one of the embodiments,.
In one of the embodiments, in the step S30, the method for the activated solid extraction column is first with after methanol
Solid-phase extraction column is activated with ultra-pure water.
In one of the embodiments, the second acetonitrile solution of various concentration described in the step S32 be successively with
The volume ratio of water is 10：90 and 50：50 acetonitrile solution.Third acetonitrile solution is and water described in the step S33
Volume ratio is 90：10 acetonitrile solution.
The collaborative detection method of beauvericin and enniatin uses Solid Phase Extraction conduct in above-mentioned grain and its product
Pretreatment technology can remove impurity, reduce matrix effect.Determinand can also be enriched with simultaneously, improves the selectivity of method.The present invention
Step needed for the pre-treating method of foundation is less, and flow is simple.The separating degree of five kinds of substances is very good, retention time and standard items
Amplitude of variation be no more than 5%, 103.9%~147.5%, the rate of recovery is higher for the recovery of standard addition of each toxin.
This method, as detection instrument, can be measured white in grain and its product simultaneously using high performance liquid chromatography tandem mass spectrum
The content of stiff rhzomorph and 4 kinds of enniatins.Have the advantages that selectivity is strong, sensitivity is good, accuracy is high and detection limit is low, be
The pollution monitoring of beauvericin and enniatin provides reliable technological means in China's grain and its product.
Description of the drawings
Fig. 1 detects chromatogram for beauvericin of the embodiment of the present invention and 4 kinds of enniatins.
Fig. 2A quantitatively detects chromatogram for enniatine B of the embodiment of the present invention.
Fig. 2 B are enniatine B qualitative detection chromatogram of the embodiment of the present invention.
Fig. 3 A quantitatively detect chromatogram for enniatine B of the embodiment of the present invention 1.
Fig. 3 B are 1 qualitative detection chromatogram of enniatine B of the embodiment of the present invention.
Fig. 4 A quantitatively detect chromatogram for enniatine A of the embodiment of the present invention 1.
Fig. 4 B are 1 qualitative detection chromatogram of enniatine A of the embodiment of the present invention.
Fig. 5 A quantitatively detect chromatogram for enniatine A of the embodiment of the present invention.
Fig. 5 B are enniatine A qualitative detection chromatogram of the embodiment of the present invention.
Fig. 6 A quantitatively detect chromatogram for beauvericin of the embodiment of the present invention.
Fig. 6 B are beauvericin qualitative detection chromatogram of the embodiment of the present invention.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, by the following examples and with reference to attached
The collaborative detection method of beauvericin and enniatin in the grain and its product of the present invention is further described in figure.
It should be appreciated that specific embodiment described herein is not intended to limit the present invention only to explain the present invention.
Referring to Fig. 1, the embodiment of the present invention provides the association of beauvericin and enniatin in a kind of grain and its product
Same detection method.Using beauvericin in Solid Phase Extraction high performance liquid chromatography tandem mass spectrum method measure grain and its product and 4 kinds
Enniatin has the advantages that pre-treatment is simple, selectivity is strong, sensitivity is good, accuracy is high and detection limit is low.
The collaborative detection method of beauvericin and enniatin includes the following steps S1 extremely in the grain and its product
S4：
S1 will be dissolved in organic solution mesoscale eddies, oscillation after grain and cereal product pulverization process and stand.Preferably,
Organic solvent is the first acetonitrile solution, it is further preferred that acetonitrile volume fraction in the first acetonitrile solution for 80%~
90%, more preferably 85%.
The Extraction solvent that grain and cereal product select in pretreatment process is particularly critical.Organic solvent preferably selects to try out
The wider reagent of property.Preferably, the first acetonitrile solution is as extraction solution.One side acetonitrile can be miscible with water, on the other hand
Acetonitrile belongs to eurytopicity Extraction solvent.Compared to the common Extraction solvent such as methanol, ethyl acetate, interference of the acetonitrile extracting solution to matrix
It is relatively small.And this five kinds of target analytes belong to the low polar substances of macromolecular, acetonitrile is preferable to its dissolubility.In an embodiment
In, the pre-treatment organic solvent of selection is identical with the organic solvent used in gradient elution in later stage Mass Spectrometer Method (being acetonitrile),
The good linking between pre-treatment and upper machine testing is realized, effect of dissolving each other is more preferable.Grain and cereal product due to its own
Particularity, the acetonitrile volume fraction of the first acetonitrile solution is 80%~90%, such as 85%, and it is right in extraction process to reduce
The degradation of target analytes.In addition, the simpler sample pretreatment process the better, target analysis in grain and cereal product can be reduced
The loss of object.And then the accuracy of sample detection is improved, and shorten analysis time.
S2 takes the supernatant after standing to be diluted with ultra-pure water.
Ultrapure water-reducible effect is to carry out concentration debugging to supernatant.Preferably, the volume ratio of ultra-pure water and supernatant
It is 1:1~1:10.
Supernatant solid phase extraction concentration after dilution is purified, and collects eluent by S3.
The step of solid phase extraction concentration described in the step S3 purifies preferably includes following steps S30 to S33：
S30 activates the solid-phase extraction column used in Solid Phase Extraction.In one embodiment, successively with methanol and ultrapure
Water activated solid extraction column.Preferably, with isometric methanol and ultra-pure water successively activated solid extraction column.Preferably, solid phase
Extraction column is C18Solid-phase extraction column.In one embodiment, more specifically, successively with 3mL methanol and the activation of 3mL ultra-pure waters with C18
Solid-phase extraction column for filler.
S31, by the solid-phase extraction column after the supernatant overactivation after being diluted in step S2.
S32 elutes the solid-phase extraction column successively with the second acetonitrile solution of various concentration.
The second acetonitrile solution of various concentration described in the step S32 is second successively in one of the embodiments,
The volume ratio of nitrile and water is 10:90 and 50:50 acetonitrile solution.
S33 elutes the solid-phase extraction column with third acetonitrile solution, it is spare to collect the eluent.
Third acetonitrile solution described in the step S33 is the volume ratio of acetonitrile and water in one of the embodiments,
It is 90：10 acetonitrile solution.
The eluent is carried out high performance liquid chromatography tandem mass spectrum detection by S4.
The step S4 includes step S41 to S42：
Eluent is carried out high performance liquid chromatography separation, mobile phase A selected as ammonium acetate solution, Mobile phase B choosing by S41
It is selected as acetonitrile solvent.Gradient elution, the gradient elution program are：At the 0th~2 minute, the volume of mobile phase A and Mobile phase B
Than 100:0；At the 2nd~3 minute, the volume ratio 40 of mobile phase A and Mobile phase B:60；At the 3rd~19 minute, mobile phase A was with flowing
The volume ratio 30 of dynamic phase B:70；At 19-21 minutes, the volume ratio 100 of mobile phase A and Mobile phase B:0.Preferably, ammonium acetate
Concentration of aqueous solution is 1mmol/L~10mmol/L, it is further preferred that a concentration of 2mmol/L of ammonium acetate solution.Implement one
In example, flow velocity 0.20mL/min, column temperature is 35 DEG C, and sample room temperature is 15 DEG C, and sample size is 5 μ L.According to separating degree, sensitive
The factors such as degree, analysis time and hangover time, to mass spectral analysis, common mobile phase composition is screened.It is final to determine to use
Ammonium acetate solution is as mobile phase A (wherein ammonium acetate can play the role of optimizing peak shape), and acetonitrile is as Mobile phase B.
Since analyte is more complicated, mobile phase elution process should not select Gradient elution.Using gradient elution analysis
Help to shorten appearance time, and effectively remove remaining impurity in chromatographic column.Gradient elution time stage by stage and stream
The ratio of dynamic phase A and Mobile phase B, larger impact is respectively provided with for the separating degree of appearance time and five kinds of target analytes.Optimization
Foundation be, in sample the retention time of target analytes compared with the retention time of target analytes in Quality Control sample, change width
Degree is no more than 5%, referring to chromatogram 1.
Selection for chromatographic column in high-efficient liquid phase chromatogram condition considers system pressure limitation by the particle size range of chromatographic column
Control is between 1 μm~3 μm, chromatogram column length range 50nm~100nm.Further, since analysis species are complicated, polarity range
It is larger, the C for being suitable for the wide polarity of separation is selected in terms of chromatographic column18Chromatographic column.Optionally, chromatographic column：Waters UPLC BEH
C18Column (2.1mm × 50mm, 1.7 μm), which employs advanced core-shell structure copolymer technology, completely porous with tradition
Silicagel column is compared, and separating degree and sensitivity all have clear improvement.
S42, to carrying out Mass Spectrometer Method into sample liquid after gradient elution.
The MS detection parameters are：Ion source is electric spray ion source (ESI sources), and scan pattern is scanned for cation, gone molten
Agent temperature is 500~600 DEG C, and ionizing voltage is 5000~6000v, and residence time is 100~300ms.
Preferably, the mass spectroscopy condition is as follows：
Ion source is electric spray ion source (ESI sources), and scan pattern is scanned for cation, and it is 550 DEG C to remove solvent temperature, from
Sonization voltage is 5500v, and gas curtain atmospheric pressure is 30psi, and collision gas is medium flow rate, and atomization gas pressure is 80psi, and auxiliary heats
Atmospheric pressure is 80psi, and residence time 200ms, detection mode is multiple-reaction monitoring.
Classification is according to target analyzed using liquid phase and dilutes hybrid standard stock solution to a concentration of 0.2mg/L, is used
Constant current injection slurry to carry out parameter optimization in the flow velocity of 5 μ g/min injection mass ion source, respectively using Q1Scan,
The scan patterns such as ProductIon and MRM determine the parent ion and daughter ion of target analytes, and using Ramp function optimizations simultaneously
Determine the parameters such as cluster voltage (DP), impact energy (CE), collision cell exit potential (CXP) and entrance potential (EP).
The mass spectrometry parameters of 5 kinds of toxin are shown in Table 1.
The mass spectrometry parameters of 15 kinds of toxin of table
Note：* it is quantitative daughter ion, DP is removes cluster voltage, and CE is impact energy, and CXP is collision cell exit potential, and EP is entrance
Voltage.
This method, as detection instrument, can be measured white in grain and its product simultaneously using high performance liquid chromatography tandem mass spectrum
The content of stiff rhzomorph and 4 kinds of enniatins.As shown in Figure 1, the chromatographic peak of five kinds of mycotoxins can be opened up on a chromatogram
Show, and without superposition, separating degree is preferable.Every group of peak is made of quota ion pair peak and qualitative ion pair peak, and quota ion pair generates
Peak it is high compared with the peak response intensity that qualitative ion pair generates in Fig. 1.This method is with selectivity is strong, sensitivity is good, accuracy
High and detection limits low advantage, and the pollution monitoring for beauvericin and enniatin in China's grain and its product provides reliably
Technological means.
The embodiment of the present invention, as pretreatment technology, can remove impurity, reduce matrix effect, simultaneously also using Solid Phase Extraction
Determinand can be enriched with, improves the selectivity of method.Step needed for the pre-treating method that the present invention establishes is less, and flow is simple.
Embodiment 1
By taking wheat and its product as an example, amount to 17 parts including wheat, flour and vermicelli, measure beauvericin and 4 kinds of grace sickles
The content of spore rhzomorph.The content of determinand in each sample is calculated using matrix matching calibration curve method.By wheat, flour and vermicelli
Grains is waited to be detected according to above-mentioned S1 to S5 steps.
It is as follows：
S1 will be dissolved in the first acetonitrile solution that acetonitrile volume fraction is 85% after wheat and its product pulverization process
It is vortexed, vibrates and stand；
S2 takes the supernatant after standing to be diluted with ultra-pure water；
Supernatant solid phase extraction concentration after dilution is purified, and collects eluent by S3；Solid phase described in the step S3
The step of extracting and enriching purifies specifically includes following steps：
S30, solid-phase extraction column C18Solid-phase extraction column；Solid-phase extraction column used in Solid Phase Extraction is activated；Successively
With 3mL methanol and the activation of 3mL ultra-pure waters with C18Solid-phase extraction column for filler.
S31, by the solid-phase extraction column after supernatant overactivation described in the 4mL after being diluted in step S2.
S32 is successively 10 with the volume ratio of acetonitrile and water:90 and 50:50 the second acetonitrile solution elutes the solid phase
Extraction column.
S33 is 90 with the volume ratio of acetonitrile and water：10 third acetonitrile solution elutes the solid-phase extraction column, collects
The eluent is spare.
Eluent is carried out high performance liquid chromatography tandem mass spectrum detection by S4, including：
S41, high performance liquid chromatography separation, mobile phase A selected as ammonium acetate solution, Mobile phase B selected as acetonitrile solvent.
Gradient elution, the gradient elution program are：At the 0th~2 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；The 2nd
~3 minutes, the volume ratio 40 of mobile phase A and Mobile phase B:60；The 3rd~19] minute, the volume ratio of mobile phase A and Mobile phase B
30:70；At the 19th~21 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；
S42, Mass Spectrometer Method.
Mass Spectrometry Conditions are：
Ion source：Electric spray ion source (ESI sources)；
Detection mode：Cation scans；
Detection mode：Multiple-reaction monitoring；
It is 550 DEG C to remove solvent temperature；
Ionizing voltage is 5500v；
Gas curtain atmospheric pressure is 30psi；
Collision gas is medium flow rate；
Atomization gas pressure is 80psi；
Auxiliary heating atmospheric pressure is 80psi；
Residence time is 200ms；
Accordingly result is obtained, as shown in Figure 1, it can be seen that the separating degree in chromatogram between adjacent peak is very good, five kinds
The chromatographic peak of mycotoxin can be shown on a chromatogram, and without superposition, separating degree is preferable, and there is quota ion pair peak at each peak
It is formed with qualitative ion pair peak, the peak that quota ion pair generates is in Fig. 1 peak, and the peak that qualitative ion pair generates is being low
Peak.High performance liquid chromatography Mass Spectrometer Method can be completed within 21 minutes.
By matrix matching standard curve, concrete content is obtained.
It the results are shown in Table 2.
The content (n=17, μ g/kg) of 5 kinds of toxin in 2 wheat of table and its product
| BEA | ENA | ENA1 | ENB | ENB1 | |
| Recall rate (%) | 100 | 100 | 100 | 100 | 100 |
| Average content (μ g/kg) | 3.57 | 1.16 | 3.64 | 14.13 | 15.10 |
| Maximum value (μ g/kg) | 4.91 | 4.61 | 20.11 | 111.95 | 71.66 |
| Minimum value (μ g/kg) | 3.31 | 0.64 | 0.58 | 1.25 | 4.54 |
| Median (μ g/kg) | 3.38 | 0.71 | 0.89 | 3.68 | 5.56 |
Wherein, recall rate is more than that the positive number of detection limit accounts for the percentage of total number of samples for content of toxins.
Embodiment 2
The present embodiment 2 is substantially the same manner as Example 1, differs only in by taking corn and its product as an example, including niblet, jade
Rice residue and corn flour amount to 19 parts, measure the content of beauvericin and 4 kinds of enniatins.Using matrix matching calibration curve method
Calculate the content of determinand in each sample.By niblet, maize pulp and corn flour sample according to it is above-mentioned be S1 to S5 steps carry out
Detection, obtains accordingly result, brings respective matrix matching standard curve into, the results are shown in Table 3.
The content (n=19, μ g/kg) of 5 kinds of toxin in 3 corn of table and its product
| BEA | ENA | ENA1 | ENB | ENB1 | |
| Recall rate (%) | 100 | 100 | 100 | 5.3 | 100 |
| Average content (μ g/kg) | 0.97 | 0.06 | 0.21 | 0.09 | 0.09 |
| Maximum value (μ g/kg) | 6.22 | 0.16 | 0.28 | 0.09 | 0.18 |
| Minimum value (μ g/kg) | 0.01 | 0.04 | 0.19 | 0.09 | 0.08 |
| Median (μ g/kg) | 0.18 | 0.04 | 0.20 | 0.09 | 0.08 |
Above example shows that the method for the present invention can be applied to different grains and its product (such as wheat and its product, corn
And its product) in the measure of beauvericin and 4 kinds of enniatin contents.
Sensitivity analysis：Using instrument signal to noise ratio as 3:1 and 10:Detection limit of the corresponding analyte concentration as method when 1
And quantitative limit.The range of linearity of 5 kinds of toxin is respectively beauvericin in the method for the present invention：0.033~100 μ g/L, grace Fusariumsp
Plain A：0.005~15 μ g/L, enniatine A1：0.013~40 μ g/L, enniatine B：0.013~40 μ g/L and grace sickle
Spore rhzomorph B1：0.033~100 μ g/L.And linearly dependent coefficient is all higher than 0.99, linear relationship is good.The detection limit of each toxin
4A and table 4B are shown in Table with quantitative limit, it is seen that the detection limit of the method for the present invention is low and high sensitivity.
The detection limit and quantitative limit of 5 kinds of toxin in table 4A different substrates
The detection limit and quantitative limit of 5 kinds of toxin in table 4B different substrates
The rate of recovery is analyzed：
Each matrix weighs a certain amount of blank sample respectively, adds in the hybrid standard liquid of 5 kinds of toxin of a certain concentration.According to step
Suddenly it is detected, obtains corresponding result A；Each matrix weighs a certain amount of blank sample respectively, by above-mentioned S1-S3 extraction and cleanings
After obtain bare substrate extracting solution, be separately added into the hybrid standard liquid of 5 kinds of toxin of a certain concentration, be configured to matrix mark-on work
Liquid is detected by above-mentioned steps S4, and accordingly result B is calculated.The calculation formula of recovery of standard addition (RE, %) is：The method of the present invention has respectively investigated the recovery of standard addition of 3 concentration levels.The result is shown in table 5, each poison
For the recovery of standard addition of element 103.9%~147.5%, the rate of recovery is higher.
The recovery of standard addition of 5 kinds of toxin in table 5A different substrates
The recovery of standard addition of 5 kinds of toxin in table 5B different substrates
Precision Analyze：
Each matrix of Precision Analyze weighs a certain amount of blank sample respectively, adds in the mixing mark of 5 kinds of toxin of known concentration
Quasi- liquid, every part of sample do 6 it is parallel.It is detected according to above-mentioned steps, the relative standard for calculating each toxin in parallel sample is inclined
Poor (RSD), evaluates the repeatability of the method for the present invention, the results are shown in Table 6.The RSD of the method for the present invention repeatability is in 1.78%2.42%
Between, show that this method has good precision.
In 6 different substrates of table during 5 kinds of toxin determinations method repeatability
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
The all possible combinations for applying each technical characteristic in example are all described.However, as long as the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (12)
1. the collaborative detection method of beauvericin and enniatin in a kind of grain and its product, this method includes following step
Suddenly：
S1 will be dissolved in organic solution mesoscale eddies, oscillation after grain and cereal product pulverization process and stand；
S2 takes the supernatant after standing to be diluted with ultra-pure water；
The supernatant solid phase extraction concentration after dilution is purified, and collects eluent by S3；
The eluent is carried out high performance liquid chromatography tandem mass spectrum detection by S4, including：
S41, high performance liquid chromatography separation, mobile phase A selected as ammonium acetate solution, Mobile phase B selected as acetonitrile solvent；Gradient
Elution, the gradient elution program are：At the 0th~2 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；At the 2nd~3 point
The volume ratio 40 of clock, mobile phase A and Mobile phase B:60；At the 3rd~19 minute, the volume ratio 30 of mobile phase A and Mobile phase B:70；
At the 19th~21 minute, the volume ratio 100 of mobile phase A and Mobile phase B:0；And
S42, Mass Spectrometer Method.
2. the collaborative detection method of beauvericin and enniatin in grain according to claim 1 and its product,
It is characterized in that：A concentration of 1mmol/L~the 10mmol/L of ammonium acetate solution.
3. the collaborative detection method of beauvericin and enniatin in grain according to claim 2 and its product,
It is characterized in that：A concentration of 2mmol/L of ammonium acetate solution.
4. the collaborative detection method of beauvericin and enniatin in grain according to claim 1 and its product,
It is characterized in that：Organic solvent described in the step S1 is the first acetonitrile solution.
5. the collaborative detection method of beauvericin and enniatin in grain according to claim 4 and its product,
It is characterized in that：The volume fraction of acetonitrile is 85% in first acetonitrile solution.
6. the collaboration inspection of beauvericin and enniatin in grain according to any one of claims 1-5 and its product
Survey method, it is characterised in that：The enniatin includes enniatine A, enniatine A1, enniatine B and grace
Fusanin B1At least one of.
7. the collaborative detection method of beauvericin and enniatin in grain according to claim 1 and its product,
It is characterized in that：The Mass Spectrometer Method condition is：
Ion source is electric spray ion source, and scan pattern is scanned for cation, and it is 500~600 DEG C to remove solvent temperature, ionization electricity
It presses as 5000~6000v, residence time is 100~300ms.
8. the collaborative detection method of beauvericin and enniatin in grain according to claim 7 and its product,
It is characterized in that：The condition of the Mass Spectrometer Method is：
Ion source is electric spray ion source, and scan pattern is scanned for cation, and it is 550 DEG C to remove solvent temperature, and ionizing voltage is
5500v, gas curtain atmospheric pressure are 30psi, and collision gas is medium flow rate, and atomization gas pressure is 80psi, and auxiliary heating atmospheric pressure is
80psi, residence time 200ms, detection mode are multiple-reaction monitoring.
9. the collaborative detection method of beauvericin and enniatin in grain according to claim 1 and its product,
It is characterized in that：The step of solid phase extraction concentration described in the step S3 purifies includes：
S30 activates the solid-phase extraction column used in Solid Phase Extraction；
S31, by the solid-phase extraction column after the supernatant overactivation after being diluted in step S2；
S32 elutes the solid-phase extraction column successively with the second acetonitrile solution of various concentration；And
S33 elutes the solid-phase extraction column with third acetonitrile solution, it is spare to collect the eluent.
10. the collaborative detection method of beauvericin and enniatin in grain according to claim 9 and its product,
It is characterized in that：Second acetonitrile solution of various concentration described in the step S32 be successively with the volume ratio of water be 10：90 Hes
50：50 acetonitrile solution；Third acetonitrile solution described in the step S33 be with the volume ratio of water be 90：10 acetonitrile
Aqueous solution.
11. the collaborative detection method of beauvericin and enniatin in grain according to claim 9 and its product,
It is characterized in that：The solid-phase extraction column is octadecylsilane chemically bonded silica.
12. the collaborative detection method of beauvericin and enniatin in grain according to claim 9 and its product,
It is characterized in that：In the step S30, the method for the activated solid extraction column for first with after methanol with ultra-pure water to Solid Phase Extraction
Column is activated.
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| CN110286178A (en) * | 2019-08-05 | 2019-09-27 | 弘正道（中国）中药研究有限公司 | The high throughput analysis method of 22 kinds of mycotoxins in a kind of detection health-care food oral liquid |
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