CN102213656B - Method for extracting and purifying and detecting type B trichothecene toxin from cereals - Google Patents
Method for extracting and purifying and detecting type B trichothecene toxin from cereals Download PDFInfo
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Abstract
The invention relates to a method for extracting and purifying and detecting type B trichothecene toxin from cereals, which aims to detect deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ACDON) and 15-acetyldeoxynivalenol (15-ACDON). The method comprises the following steps of: adding isopyknic absolute ethanol into water extract solution of a sample to be detected, precipitating to remove most of proteins and polysaccharides, rotating at low temperature to evaporate supernate to dryness, adding acetonitrile-Nacl aqueous solution, demixing an organic phase and an aqueous phase, and removing the aqueous phase to remove strongly-polar impurities; and rotating and evaporating the organic phase to dryness, dissolving the organic phase in water, purifying by an OASIS solid phase extraction column, activating, coating the sample, analyzing and the like, performing high-performance liquid chromatography-ultraviolet (HPLC-UV) detection on the prepared sample, and analyzing the DON, the 3-ACDON and the 15-ACDON which serve as reference samples qualitatively and quantitatively. In the method, impurity peaks do not exert interference with the detected samples, and the 3-ACDON and the 15-ACDON which are geometric isomers have high separation degree.
Description
Technical field
The present invention relates to the extraction purification assays method of Type B trichothecene family toxin in the cereal, belong to the analytical test field.
Background technology
By the microbial head blight of reaping hook is one of very important disease on the cereal crop, has had a strong impact on world food safety.Fusarium toxin is one type of secondary metabolite that sickle-like bacteria produces.Kind of compound surplus trichothecene family toxin includes 200 is in the fusarium toxin very important one type.Deoxynivalenol (deoxynivalenol, DON), nivalenol (Nivalenol, NIV) and derivant belong to trichothecene family category-B, basic structure is the sequiterpene at Fourth Ring, is characteristic with the carbonyl on the C8 position.Wherein, deoxynivalenol (DON) occupy cereal pollution rate and level of pollution first of the fusarium toxin, causes grave danger for the health of people, domestic animal.Miller is different according to the acetylizad position of DON; The chemical type of DON is divided into 3-acetyl deoxynivalenol (3-acetyldeoxynivalenol again; 3-ACDON) with 15-acetyl deoxynivalenol (15-acetyldeoxynivalenol, 15-ACDON).
Trichothecene same clan compound is comparatively heat-resisting, needs to surpass 200 ℃ and just can be destroyed, and is also more stable to bronsted lowry acids and bases bronsted lowry, therefore, and through cooking difficult processing usually to destroy its activity.The food of pollution mainly comprises cereal such as corn, wheat, rye, oat.Detect trichothecene family toxin such as DON and in grain, have situation, and monitor, the safety of underwriter's based food is very important.
The detection method of fusarium toxin is more, can be divided into 3 types: 1. bioassay method comprises skin toxicology test, seed germination experiment etc.; 2. the physics and chemistry detection method comprises thin-layer chromatography, high performance liquid chromatography, gas chromatography, the coupling of gas (liquid) matter etc.; 3. the immunochemistry detection method comprises radioimmunoassay method, fluorescence polarization immunoassay and enzyme linked immunological absorption (ELISA) method.Wherein, high performance liquid chromatography is wide to the application of sample, does not receive the restriction of analytic target volatility and thermal stability; And be widely used; Detect Type B trichothecene family toxin and be mostly to detect with the cumarin fluorescence detector of deriving, assorted peak disturbs few, but needs the loaded down with trivial details step of deriving.When using UV-detector to detect; Grain samples for natural pollution; After multiple function and immune affinity column purify, toxin occurs, once reported like Klotzel etc. and use current wheat samples purification test experience of carrying out decontaminating column that commercialization produces commonly used on the market, comprise two kinds of immune affinity columns and based on activated charcoal and aluminium oxide pillar clean-up effect as the MycoSep multiple function of filler to natural pollution with the grain base matter extracts altogether, the problem of spectrogram degree of separation difference; Experimental result shows; Purify the back through these three kinds decontaminating columns commonly used and detect with UV-detector, impurity peaks is more, receives matrix interference serious.There is the non-specific interaction that extracts altogether with the grain base matter in toxin, peak that extracts altogether and toxin signal overlap, covering, and also the HPLC chromatographic column will each EOS post-flush, and guard column also must often be replaced.
Summary of the invention
The objective of the invention is to existing impurity peaks more in the UV-detector detection; The separation and purification and the detection method of a kind of new Type B trichothecene family toxin that receives problems such as matrix interference is serious and provide are primarily aimed at the detection of DON, 3-ACDON, 15-ACDON.
The present invention is achieved in that described a kind of extraction purification assays method to Type B trichothecene family toxin in the cereal; It is characterized in that: at first tested cereal sample is pulverized back water vibration and extract; Then extract is carried out purifying; Adopt the HPLC-UV method to detect again, can carry out qualitative and quantitative analysis the Type B trichothecene in the cereal sample.
In the present invention, described method for extraction and purification to Type B trichothecene family toxin in the cereal is characterized in that: take by weighing cereal sample 5.0g after the pulverizing in the centrifuge tube of 50ml, add the H of 25ml
2The O vortex oscillation is extracted 30min, and extract is used the Buchner funnel suction filtration, gets 10ml filtrating and adds 4 ℃ of depositions of isopyknic absolute ethyl alcohol 2h, and centrifugal 10min abandons deposition under 10000rpm, gets supernatant rotation evaporate to dryness, adds 15ml acetonitrile-H
2O, acetonitrile and H
2The volume ratio of O is 2: 1, adds NaCL again, static acetonitrile and H after the vortex oscillation
2The O layering is heated 2min in 80 ℃ of water-baths, abandon water after the cooling, adds anhydrous sodium sulfate dehydration to acetonitrile in mutually, gets the 5ml acetonitrile and rotates evaporate to dryness, 0.5mlH mutually
2The O extraction of vibrating at twice merges extract, is that the Waters OASISHLB solid phase extraction column of 3ml/60mg is further purified through specification; Requirement according to the pillar operation instructions is carried out activation and last appearance to pillar, resolves natural flow velocity with the ethyl acetate of 1ml then; Obtain desorbed solution, desorbed solution is rotated evaporate to dryness after, separate with 0.5ml is water-soluble; Cross 0.22 μ m miillpore filter at last, last machine is to be measured.
In the present invention, described HPLC-UV detection method to Type B trichothecene family toxin in the cereal is characterized in that: chromatographic column: C18; Moving phase: A acetonitrile, the phosphate aqueous solution of B 0.001%; The gradient elution program is: 0～6min, 92.5～90.0% acetonitriles; 6～18min, 90%～60% acetonitrile; 18～22min, 60% acetonitrile; 22～25min, 60%～92.5% acetonitrile; 25～35min, 92.5% acetonitrile; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Sample size: 15 μ l; UV-detector: 224nm.
In the present invention, described purification assays method to Type B trichothecene family toxin in the cereal, described cereal is meant: corn or wheat class.
In the present invention, described qualitative and quantitative analysis to Type B trichothecene family toxin in the cereal is meant: the DON in the Type B trichothecene family toxin in the tested cereal sample or 3-ACDON or 15-ACDON are carried out qualitative and quantitative analysis.
In the present invention; Described qualitative and quantitative analysis to Type B trichothecene family toxin in the cereal is meant: in high-efficient liquid phase chromatogram; Standard specimen DON, 3-ACDON and 15-ACDON and the tested cereal sample of Type B trichothecene family toxin are compared; Appearance time according to standard specimen is qualitative, carries out quantitative test according to peak area.
The invention has the advantages that: it is few that the method for extraction and purification that is adopted disturbs the assorted peak of collection of illustrative plates, need not often to change guard column, can save loaded down with trivial details derivatization operation steps; Can detect DON, 3-ACDON, 15-ACDON in the Type B trichothecene family toxin simultaneously.Through optimizing moving phase isochromatic spectrum condition, under the condition of not deriving, can make isomers 3-ACDON and 15-ACDON that degree of separation is preferably arranged; Though it is bigger that gradient elution program A, B phase ratio fluctuate, baseline is steady.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of wheat samples 1 and standard items;
Fig. 2 is the high-efficient liquid phase chromatogram of wheat samples 2 and standard items.
Embodiment
Through embodiment the present invention is carried out concrete description below; Be necessary to be pointed out that at this following examples only are used for the present invention is further explained; But can not be interpreted as the restriction to protection domain of the present invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment 1
1, the selection of standard specimen:
Standard specimen is purchased in Sigma-Aldrich company (down together).
2, the method for extraction and purification of Type B trichothecene family toxin in the wheat:
(1) specimen preparation: No. 13 wheat samples 5.0g of peaceful wheat that take by weighing the results in 2010 after the pulverizing add the H of 25ml in the centrifuge tube of 50ml
2The O vortex oscillation is extracted 30min, and extract is used the Buchner funnel suction filtration, gets 10ml filtrating and adds 4 ℃ of depositions of isopyknic absolute ethyl alcohol 2h, and centrifugal 10min abandons deposition under 10000rpm, gets supernatant rotation evaporate to dryness, adds 15ml acetonitrile-H
2O (2: 1, V/V), add NaCL again, static acetonitrile and H after the vortex oscillation
2The O layering is heated 2min in 80 ℃ of water-baths, abandon water after the cooling, adds anhydrous sodium sulfate dehydration to acetonitrile in mutually, gets the 5ml acetonitrile and rotates evaporate to dryness mutually, 0.5ml H
2The O extraction of vibrating at twice merges extract, is carrying out OASIS solid phase extraction column purifying:
(2) the use step of OASIS solid phase extraction column is following:
A. the activation of pillar: after the drip washing of 2ml methyl alcohol, add 2ml H again
2O drip washing is the nature flow velocity, and vacuum is drained afterwards; B. go up appearance: applied sample amount is 1ml, natural flow velocity, and vacuum is drained 30min afterwards; C. resolve: the ethyl acetate of 1ml is resolved, natural flow velocity.After desorbed solution rotated evaporate to dryness, separate with 0.5ml is water-soluble, cross 0.22 μ m miillpore filter, last machine is to be measured.
3, adopt HPLC-UV to detect its testing conditions:
Chromatographic column: C18 (250mm * 4.6,5 μ m); Moving phase: A acetonitrile, the phosphate aqueous solution of B 0.001%; Gradient: 0～6min, 92.5～90.0% acetonitriles; 6～18min, 90%～60% acetonitrile; 18～22min, 60% acetonitrile; 22～25min, 60%～92.5% acetonitrile; 25～35min, 92.5% acetonitrile; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Sample size: 15 μ l; UV-detector: 224nm.
4, atlas analysis
Visible by Fig. 1:
In standard specimen: standard items purity is respectively DON >=99.4%, 3-ACDON >=98%, 15-ACDON >=98.8% (purchasing in Sigma-Aldrich).The appearance time of DON, 15-ACDON and 3-ACDON is respectively in the collection of illustrative plates: 13.94min, 19.86min, 20.17min.
In sample 1: detect DON, content is 498 μ g/kg, meets China's limit standard 1000 μ g/kg, does not detect 15-ACDON and 3-ACDON, proves that No. 13 these a collection of wheat seeds of peaceful wheat of results in 2010 are edible.
Embodiment 2
1, the method for extraction and purification of Type B trichothecene family toxin in the wheat:
(1) specimen preparation: No. 11 wheat samples 5.0g of peaceful wheat that take by weighing the results in 2007 after the pulverizing add the H of 25ml in the centrifuge tube of 50ml
2The O vortex oscillation is extracted 30min, and extract is used the Buchner funnel suction filtration, gets 10ml filtrating and adds 4 ℃ of depositions of isopyknic absolute ethyl alcohol 2h, and centrifugal 10min abandons deposition under 10000rpm, gets supernatant rotation evaporate to dryness, adds 15ml acetonitrile-H
2O (2: 1, V/V), add NaCL again, static acetonitrile and H after the vortex oscillation
2The O layering is heated 2min in 80 ℃ of water-baths, abandon water after the cooling, adds anhydrous sodium sulfate dehydration to acetonitrile in mutually, gets the 5ml acetonitrile and rotates evaporate to dryness mutually, 0.5ml H
2The O extraction of vibrating at twice merges extract, is carrying out OASIS solid phase extraction column purifying:
(2) the use step of OASIS solid phase extraction column is following:
A. the activation of pillar: after the drip washing of 2ml methyl alcohol, add 2ml H again
2O drip washing is the nature flow velocity, and vacuum is drained afterwards; B. go up appearance: applied sample amount is 1ml, natural flow velocity, and vacuum is drained 30min afterwards; C. resolve: the ethyl acetate of 1ml is resolved, natural flow velocity.After desorbed solution rotated evaporate to dryness, separate with 0.5ml is water-soluble, cross 0.22 μ m miillpore filter, last machine is to be measured.
2, adopt HPLC-UV to detect its testing conditions:
Chromatographic column: C18 (250mm * 4.6,5 μ m); Moving phase: A acetonitrile, the phosphate aqueous solution of B 0.001%; Gradient: 0～6min, 92.5～90.0% acetonitriles; 6～18min, 90%～60% acetonitrile; 18～22min, 60% acetonitrile; 22～25min, 60%～92.5% acetonitrile; 25～35min, 92.5% acetonitrile; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Sample size: 15 μ l; UV-detector: 224nm.
3, atlas analysis
Visible by Fig. 2:
In standard specimen: standard items purity is respectively DON >=99.4%, 3-ACDON >=98%, 15-ACDON >=98.8% (purchasing in Sigma-Aldrich).The appearance time of DON, 15-ACDON and 3-ACDON is respectively in the collection of illustrative plates: 13.94min, 19.86min, 20.17min.
In sample 2: detect DON; Content is 1248 μ g/kg, surpasses China's limit standard 1000 μ g/kg, explains that No. 11 wheat seeds of this a collection of peaceful wheat of results in 2007 cannot eat; Simultaneously, the content that also detects 15-ACDON and 3-ACDON is respectively 182 μ g/kg, 96 μ g/kg.
Claims (5)
1. extraction purification assays method to Type B trichothecene family toxin in the cereal; It is characterized in that: at first tested cereal sample is pulverized back water vibration and extract; Then extract is carried out purifying; Adopt the HPLC-UV method to detect again, can carry out qualitative and quantitative analysis the Type B trichothecene family toxin in the cereal sample;
After tested cereal sample pulverized, the water vibration was extracted and extract is carried out purifying and is meant: take by weighing cereal sample 5.0g after the pulverizing in the centrifuge tube of 50ml, add the H of 25ml
2The O vortex oscillation is extracted 30min, and extract is used the Buchner funnel suction filtration, gets 10ml filtrating and adds 4 ℃ of depositions of isopyknic absolute ethyl alcohol 2h, and centrifugal 10min abandons deposition under 10000rpm, gets supernatant rotation evaporate to dryness, adds 15ml acetonitrile-H
2O, acetonitrile and H
2The volume ratio of O is 2: 1, adds NaCl again, leaves standstill acetonitrile and H after the vortex oscillation
2The O layering is heated 2min in 80 ℃ of water-baths, abandon water after the cooling, adds anhydrous sodium sulfate dehydration to acetonitrile in mutually, gets the 5ml acetonitrile and rotates evaporate to dryness mutually, 0.5ml H
2The O extraction of vibrating at twice merges extract, is that the Waters OASIS HLB solid phase extraction column of 3ml/60mg is further purified through specification; Requirement according to the pillar operation instructions is carried out activation and last appearance to pillar, resolves natural flow velocity with the ethyl acetate of 1ml then; Obtain desorbed solution, desorbed solution is rotated evaporate to dryness after, separate with 0.5ml is water-soluble; Cross 0.22 μ m miillpore filter at last, last machine is to be measured.
2. the extraction purification assays method to Type B trichothecene family toxin in the cereal according to claim 1 is characterized in that: chromatographic column: C18; Moving phase: A acetonitrile, the phosphate aqueous solution of B 0.001%; The gradient elution program is: 0～6min, 92.5～90.0% acetonitriles; 6～18min, 90%～60% acetonitrile; 18～22min, 60% acetonitrile; 22～25min, 60%～92.5% acetonitrile; 25～35min, 92.5% acetonitrile; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Sample size: 15 μ l; UV-detector: 224nm.
3. the extraction purification assays method to Type B trichothecene family toxin in the cereal according to claim 1, it is characterized in that: described cereal is meant: corn or wheat class.
4. according to the described extraction purification assays method to Type B trichothecene family toxin in the cereal of one of claim 1～3, it is characterized in that: described qualitative and quantitative analysis to Type B trichothecene family toxin in the cereal is meant: the DON in the Type B trichothecene family toxin in the tested cereal sample or 3-ACDON or 15-ACDON are carried out qualitative and quantitative analysis.
5. the extraction purification assays method to Type B trichothecene family toxin in the cereal according to claim 4; It is characterized in that: describedly DON in the Type B trichothecene family toxin in the tested cereal sample or 3-ACDON or 15-ACDON are carried out qualitative and quantitative analysis be meant: in high-efficient liquid phase chromatogram; DON, 3-ACDON and 15-ACDON standard specimen and the tested cereal sample of Type B trichothecene family toxin are compared; Appearance time according to standard specimen is qualitative, carries out quantitative test according to peak area.
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| CN102507774B (en) * | 2011-10-31 | 2013-10-16 | 江苏省农业科学院 | Extracting, purification and detection method for nivalenol in grains |
| CN102401820A (en) * | 2011-11-21 | 2012-04-04 | 江苏省农业科学院 | Method for detecting content of mycotoxins in wheat |
| CN102590380A (en) * | 2012-02-10 | 2012-07-18 | 江苏省农业科学院 | Method for detecting content of combined deoxynivalenol in cereal |
| CN103539841A (en) * | 2012-07-12 | 2014-01-29 | 重庆乾泰生物医药有限公司 | Separation and purification method of cyclohexanol peptide compound and salt thereof |
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| CN102297904A (en) * | 2010-06-25 | 2011-12-28 | 中国医学科学院药用植物研究所 | Method for detecting two A-type trichothecene toxins in traditional Chinese medicines with different matrixes |
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Application publication date: 20111012 Assignee: Jiangsu Zhongjiang Seed Industry Co.,Ltd. Assignor: Jiangsu Academy of Agricultural Sciences Contract record no.: 2015320000598 Denomination of invention: Method for extracting and purifying and detecting type B trichothecene toxin from cereals Granted publication date: 20120912 License type: Exclusive License Record date: 20150924 |
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