CN106153785A - A kind of online sample introduction of aflatoxin analyzes method - Google Patents
A kind of online sample introduction of aflatoxin analyzes method Download PDFInfo
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- CN106153785A CN106153785A CN201510167083.8A CN201510167083A CN106153785A CN 106153785 A CN106153785 A CN 106153785A CN 201510167083 A CN201510167083 A CN 201510167083A CN 106153785 A CN106153785 A CN 106153785A
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Abstract
The open a kind of online sample introduction of aflatoxin of the present invention analyzes method, comprise the following steps: sample extracting solution is through filtering, after dilution, filtrate is through the immune affinity column containing aflatoxin specific antibody, this antibody and AFB1, B2, G1, G2 occurs specific antigen-antibody to combine, retain it on this affinity column, other impurities with flow flow out mutually, antigen-antibody bond fission is made by heating, with water elution target compound, eluent by full-automatic mycotoxin pre-treatment and the online online sample introduction of sample introduction platform to high performance liquid chromatograph, aflatoxin is sufficiently separated, target compound derives through post-column derivation, can be detected by fluorescence detector, each aflatoxin can respectively obtain the most quantitative.This method makes antigen-antibody bond fission by heating, with water elution target compound, it is to avoid the use of organic solvent, environmental protection；Full automatic working, had both improve production efficiency and repeatability, avoided again operator to contact the aflatoxin standard substance of severe toxicity.
Description
Technical field
The present invention relates to a kind of method detecting aflatoxin, particularly a kind of full-automatic online sample introduction analysis
Method.
Background technology
Mycotoxin is the secondary metabolite of Toxigenic fungi secretion.Aflatoxin (Aflatoxin) is toxicity
The strongest mycotoxin, being delimited by the Agency for Research on Cancer of World Health Organization (WHO) (WHO) is a class carcinogen.
The hazardness of aflatoxin is that people and animal livers tissue are had destruction, may result in hepatocarcinoma time serious
Even dead.In the food of natural contamination the most common with AFB1, its toxicity and carcinogenecity are also
The strongest.Aflatoxin is widely present in agricultural product and some meat productss such as rice, Semen Maydis, Semen arachidis hypogaeae, Semen sojae atricolor
In, all define the AFB1 maximum allowable content in food and feedstuff for these countries in the world and make
For mandatory standard.Therefore the fast determining method of aflatoxin in food accurately and effectively is developed, to strengthening food
Product security control is most important.
Existing Determination Methods of Aflatoxins includes thin layer chromatography, immune analysis method and instrumental method.
Wherein thin layer chromatography is the most frequently used detection method, and common laboratory all can be carried out, but reagent dosage is big,
Complex operation, impurity serious interference, cannot accurate quantification, and bigger to experimenter and environmental pollution.Exempt from
Epidemiology is analyzed method and is included enzyme linked immunosorbent assay, radioimmunology and time-resolved fluorescence method.Enzyme linked immunological
Absorption method high specificity, highly sensitive, low cost, the batch detection that is suitable to, but be only capable of detecting single toxin (as
AFB1), and easily occur that false positive results is difficult to control to.There is radiocontamination in radioimmunology,
Now apply less.Time-resolved fluorescence method detection sensitivity is higher than the above two, but complex pretreatment, required instrument
Device is expensive, is only limitted to medical clinic applications field and uses.Instrumental method includes fluorescence spectrophotometry and height
Effect liquid phase chromatogram method, it is highly sensitive, and accuracy is good, but requires that aflatoxin sample purity is high, sample
Pretreatment process is loaded down with trivial details, the longest, it is difficult to realize quickly detection.
Summary of the invention
It is an object of the invention to provide one overcome the length loaded down with trivial details, time-consuming of sample pre-treatments in prior art,
Manual operation error is big, use severe toxicity mark product and multiple toxic organic solvents to poison operator in preprocessing process
The online sample introduction of full-automatic aflatoxin of the problem of member and pollution environment analyzes method.
For realizing object above, the open techniques below scheme of the present invention: a kind of online sample introduction of aflatoxin divides
Analysis method, it is characterised in that comprise the following steps: sample extracting solution is after filtering, diluting, and filtrate is passed through
Containing the immune affinity column of aflatoxin specific antibody, this antibody and AFB1, B2, G1,
G2 occurs specific antigen-antibody to combine so that it is being retained on this affinity column, other impurities with flow flow mutually
Going out, make antigen-antibody bond fission by heating, with water elution target compound, eluent is by full-automatic true
Verticillium toxin pre-treatment and the online online sample introduction of sample introduction platform to high performance liquid chromatograph, AFB1,
B2, G1, G2 are sufficiently separated, and target compound derives through post-column derivation, can be examined by fluorescence
Survey device detects, and each aflatoxin can respectively obtain the most quantitative.
As a preferred version, described immune affinity column volume is 0.2-0.8mL.
As a preferred version, described post-column derivation derives based on ultraviolet light.
It is an advantage of the current invention that: the aflatoxin online sample introduction analytical that (1) present invention provides
Speed is fast, and sample purification and sample introduction analysis can be carried out simultaneously, and average each sample only needs 9 minutes, and existing
Technology quantitative analysis to be accomplished needs several hours to several days time；(2) detection range is wide, highly sensitive, minimum
Detection limit (in terms of AFB1) is as 5ppt；(3) monoclonal antibody immunity technology is used, can be special
Aflatoxin and other mycotoxins are separated by opposite sex ground, accuracy, highly reliable, and the response rate is high
Reach more than 80%；(4) immune affinity column column volume is little, and solvent load is few, is greatly shortened sample processing time；
(5) antigen-antibody bond fission is made by heating, with water elution target compound, it is to avoid making of organic solvent
With, environmental protection；(6) full automatic working, round-the-clock running, on the one hand improve production efficiency and repetition
Property, on the other hand avoid operator to contact the aflatoxin standard substance of severe toxicity；(7) this system uses and adds
Aflatoxin is used pure water to elute from immune affinity column by hot mode, can directly divide by feed liquor phase system
Analysis, and tradition Aspergillus flavus toxin immuno-affinity column can not carry out heating eluting, can only be by pure organic solvent such as first
Alcohol carries out eluting, is so can not directly feed liquor facies analysis, it is necessary to hand sampling, it is impossible to realize automatically point
Analysis, needs to waste analysis time.
Accompanying drawing explanation
Fig. 1 is aflatoxin regression equation and correlation coefficient.
Fig. 2 is that mark-on reclaims result.
Fig. 3 be aflatoxin standard substance chromatogram (RP C18,250 × 4.6mm × 5um,
0.05ng/10mL), peak sequence is followed successively by AFG 2, G1, B2, B1.
Fig. 4 is AFG 2, G1, B2, B1 standard working curve.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Experiment side used in following embodiment
Method if no special instructions, is conventional method.Material used in following embodiment, reagent etc., as without special
Different explanation, the most commercially obtains.Should be understood that these embodiments be merely to illustrate the present invention and need not
In limiting the scope of the present invention.
Embodiment 1. uses full-automatic mycotoxin pre-treatment and online sample introduction platform and HPLC to detect food
Middle Aspergillus flavus poison
1, reagent and instrument and equipment
1.1 preparation of reagents
1.1.1 sample extraction solvent: methanol/water (4/1, v/v).
1.1.2 5mmol phosphate buffered solution (PBS) (pH7.2) preparation:
1.1.2.1 50.14gNa is weighed2HPO4.12H2O is dissolved in 700ml water；
1.1.2.2 9.66gNaH is weighed2PO4.1H2O is dissolved in 350ml water；
1.1.2.3 carry out being mixed and added into 42.5gNaCl by above two solution, be configured to pH7.25mmol
Phosphate buffered solution.
1.1.3 1mmol phosphate buffered solution (PBS) (pH7.2) preparation:
The 5mmol phosphate buffered solution (pH7.2) taking 200ml is diluted in 800ml water.
1.1.4 methanol/water: 40/60.
1.1.5 eluent: take solvent orange 2 A 10mL, joins inside 500mL distilled water, mixing.
1.1.6 HPLC flowing preparation mutually: water/methanol/acetonitrile (55/30/15, v/v).
1.1.7 11.2% methanol/PBS solution preparation:
Take 28mL methanol, with the PBS buffer solution constant volume of 1mmol to 250mL, mixing.
1.1.8 aflatoxin standard solution: AFG 2:0.288ug/mL；G1:1.056ug/mL；
B2:0.314ug/mL；B1:1.039ug/mL.
1.2 instrument and equipment
(1) full-automatic mycotoxin pre-treatment and online sample introduction platform.
Full-automatic mycotoxin pre-treatment and online sample introduction platform are that Shanghai huge rock conjunction scientific instrument share has
The commercially available prod of limit company, has the introduction of its product information in company's site.This platform is with full-automatic solid
The multiple combinations such as extraction mutually, gel purification, on-line/off-line accurate quantification concentration, are applicable not only to common sample
Product pre-treatment is more suitable for aflatoxin specialty pre-treatment.Can be by the most online to sample pre-treatments and HPLC
Use, make pre-treatment and analyze full-automation.
Full-automatic mycotoxin pre-treatment and online sample introduction platform include: 1, fixed support, 2, decontaminating column grabs
Take device, 3, decontaminating column heater, 4, high-pressure liquid phase injection valve be equipped with 700ul rustless steel quantitative loop.Purify
Post heater can heating-up temperature be 100 DEG C, and high-pressure liquid phase injection valve is two six-way valves.
(2) HPLC system, band fluorescence detector (FLD)
(3) UVE post-column derivatization system
(4) the affine decontaminating column of Aflaclean smart immunity
(5) high-speed homogenization machine
(6) high speed centrifuge
(7) 50mL centrifuge tube
2, experimental procedure
2.1 HPLC reference analysis method:
Aflatoxin HPLC analyzes testing conditions and can refer to following setting:
Flowing phase: water/methanol/acetonitrile 55/30/15 (v/v)；Flow velocity: 1.2ml/min；Column temperature: 36 DEG C；
HPLC analytical column: RP C18,250 × 4.6mm × 5um；Or LCTech dedicated columns 150 × 4.6mm
×5um；
Fluorescence detector is arranged: excitation wavelength EX.:365nm；Launch wavelength Em.:460nm (to use
UVE post-column derivation).
3, sample treatment
For the sample of different substrates, pre-treating method is different.
3.1 method one: standard method
This processing routine does not interferes be applicable to sample substrate, is primarily adapted for use in the place of most of frumentum sample
Reason.
Weigh 2.0g sample in 50mL centrifuge tube, and add the sodium chloride of 0.2g.It is initially charged 10mL
Extraction solvent (methanol/water, 4/1, v/v), then high-shear homogenizer homogenizing extraction 5min.By centrifuge tube at height
Speed centrifuge is centrifuged, rotating speed 8000r/min.Take supernatant 4.2mL, add 25.8mL PBS buffering molten
Liquid (pH 7.2).After stirring, centrifugal.Take supernatant about 12mL to Freestyle sample injection bottle (16mL)
In, examination with computer.
3.2 methods two: standard method adds normal hexane layering
This sample treatment is for the most containing oils and fats in sample, such as nuts, and some spice or without flower
Fruit jam, needs the impurity with normal hexane removal containing some in fat and sample.
Weigh 2.0g sample in 50mL centrifuge tube, and add the sodium chloride of 0.2g.It is initially charged 10mL
Extraction solvent (methanol/water, 4/1, v/v), adds 5mL normal hexane, then high-shear homogenizer homogenizing extracts
5min.Centrifuge tube is centrifuged in high speed centrifuge, rotating speed 8000r/min.Take off a layer aqueous phase solution 4.2mL,
Add 25.8mL PBS buffer solution (pH 7.2).Fully after mixing, centrifugal.Take supernatant 12mL left
Right in Freestyle sample injection bottle (16mL), examination with computer.
4 standard curves
Use methanol that aflatoxin standard solution is diluted 50 times, it may be assumed that AFG 2:
5.76ng/mL；G1:21.12ng/mL；B2:6.28ng/mL；B1:20.78ng/mL, this solution is made
For standard solution.
Standard curve concentration (represents with B1 concentration): 0.05ng/10mL, 0.1ng/10mL, 0.2ng/10mL,
0.4ng/10mL, 0.8ng/10mL, taking concentration the most respectively is standard solution, 12.5uL, 25uL, 50uL,
In 100uL, 200uL to 50mL volumetric flask, with 11.2% methanol/PBS buffer solution constant volume, both must go up
State standard curve.
Take in above-mentioned solution about 12mL to 16mL sample bottle and carry out examination with computer.
5 examination with computers
5.1 standard curves, at this, full-automatic mycotoxin pre-treatment is online with HPLC with online sample introduction platform
In test aflatoxin experiment, Aflaclean smart immunity is also gone up in the making of standard curve as requested
The laggard HPLC of affinity column is analyzed.
The aflatoxin assay method that sample sets according to instrument is tested by 5.2, simultaneously HPLC note
Record signal.
6, sample test and recovery testu
6.1 sample tests: after wheat samples is pulverized with pulverizer, operate according to 3.1 methods one.
Take Oleum Arachidis hypogaeae semen sample, operate according to 3.2 methods two.
6.2 sample mark-on tests: add 25uL standard in 6.1 in load weighted sample and use solution, i.e.
AFG 2:0.144ng；G1:0.528ng；B2:0.157ng；B1:0.520ng；6.1
In load weighted sample add 75uL standard use solution, i.e. AFG 2:0.432ng；G1:
1.584ng；B2:0.471ng；B1:1.56ng.
7 results
7.1 standard curves: using the method Criterion curve, the range of linearity exists
0.05ng/10mL～0.8ng/10mL, linearly dependent coefficient is all more than 0.999.
It is aflatoxin regression equation and correlation coefficient that result sees Fig. 1, Fig. 1.
7.2 recovery of standard addition
In blank sample, carry out mark-on result of the test show, the method response rate all 80%-99.4% it
Between.
Result sees Fig. 2, Fig. 2 position mark-on and reclaims result.
The above is only the preferred embodiment of the present invention, it is noted that common for the art
Technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these
Improvements and modifications also should be regarded as protection scope of the present invention.
Claims (3)
1. the online sample introduction of aflatoxin analyzes method, it is characterised in that comprise the following steps: sample
Extracting solution is after filtering, diluting, and filtrate is affine through the immunity containing aflatoxin specific antibody
Post, this antibody and AFB1, B2, G1, G2 generation specific antigen-antibody combines,
Retaining it on this affinity column, other impurities with flow flow out mutually, make antigen-antibody by heating
Bond fission, with water elution target compound, eluent by full-automatic mycotoxin pre-treatment and
The online sample introduction of line sample introduction platform is to high performance liquid chromatograph, AFB1, B2, G1, G2
Being sufficiently separated, target compound derives through post-column derivation, can be examined by fluorescence detector
Surveying, each aflatoxin can respectively obtain the most quantitative.
A kind of online sample introduction of aflatoxin the most according to claim 1 analyzes method, it is characterised in that
Described immune affinity column volume is 0.2-0.8mL.
A kind of online sample introduction of aflatoxin the most according to claim 1 analyzes method, it is characterised in that
Described post-column derivation derives based on ultraviolet light.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693273A (en) * | 2018-06-14 | 2018-10-23 | 上海上药华宇药业有限公司 | Aflatoxin B1, the detection method of B2, G1, G2 in a kind of Chinese medicine |
| CN108845056A (en) * | 2018-08-02 | 2018-11-20 | 山东省农业科学院农业质量标准与检测技术研究所 | A kind of on-line automatic pretreating device of aflatoxin based on flow path switching |
| CN110095549A (en) * | 2019-06-12 | 2019-08-06 | 吉林省产品质量监督检验院(吉林省农产品认证中心) | The detection device and method of aflatoxin in a kind of Grain and its product |
| CN110749691A (en) * | 2019-12-23 | 2020-02-04 | 山东畜牧兽医职业学院 | HPLC-MS/MS method for determining aflatoxin and homologue thereof in infant auxiliary food |
| CN112362607A (en) * | 2018-11-29 | 2021-02-12 | 平顶山市畜产品质量安全监测中心 | Detection cabinet for aflatoxin in corn feed stored in feedlot |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673746A (en) * | 2005-04-15 | 2005-09-28 | 中国农业科学院油料作物研究所 | Fast detecting method for aftatoxin B1 |
| CN101109735A (en) * | 2007-05-14 | 2008-01-23 | 劲牌有限公司 | Fluorescence photometry for immune affinity column of aflatoxin in paddy |
| CN101241128A (en) * | 2008-03-18 | 2008-08-13 | 上海大学 | Aspergillus flavus toxin immuno-affinity column preparation method |
| CN102621340A (en) * | 2012-04-16 | 2012-08-01 | 北京莱伯泰科仪器有限公司 | Full-automatic aflatoxin analytic system |
| CN104280503A (en) * | 2014-10-29 | 2015-01-14 | 中国烟草总公司湖北省公司 | HPLC-FLD pre-column derivatization method for simultaneously determining aflatoxins B1, B2, G1 and G2 in tobacco |
-
2015
- 2015-04-09 CN CN201510167083.8A patent/CN106153785A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1673746A (en) * | 2005-04-15 | 2005-09-28 | 中国农业科学院油料作物研究所 | Fast detecting method for aftatoxin B1 |
| CN101109735A (en) * | 2007-05-14 | 2008-01-23 | 劲牌有限公司 | Fluorescence photometry for immune affinity column of aflatoxin in paddy |
| CN101241128A (en) * | 2008-03-18 | 2008-08-13 | 上海大学 | Aspergillus flavus toxin immuno-affinity column preparation method |
| CN102621340A (en) * | 2012-04-16 | 2012-08-01 | 北京莱伯泰科仪器有限公司 | Full-automatic aflatoxin analytic system |
| CN104280503A (en) * | 2014-10-29 | 2015-01-14 | 中国烟草总公司湖北省公司 | HPLC-FLD pre-column derivatization method for simultaneously determining aflatoxins B1, B2, G1 and G2 in tobacco |
Non-Patent Citations (4)
| Title |
|---|
| LCTECH: "《Aflatoxin B/G in Pumpkin Seeds》", 31 October 2014 * |
| LCTECH: "《SMART Columns and FREESTYLE ThermELUTE the Special Combination for the Mycotoxin Analysis》", 31 July 2014 * |
| N. ALI ET AL.: "Evaluation of a method to determine the natural occurrence of aflatoxins in commercial traditional herbal medicines from Malaysia and Indonesia", 《FOOD AND CHEMICAL TOXICOLOGY》 * |
| 张雪辉 等: "免疫亲合柱净化HPLC柱后溴衍生化方法检测中药中黄曲霉毒素", 《中国中药杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693273A (en) * | 2018-06-14 | 2018-10-23 | 上海上药华宇药业有限公司 | Aflatoxin B1, the detection method of B2, G1, G2 in a kind of Chinese medicine |
| CN108845056A (en) * | 2018-08-02 | 2018-11-20 | 山东省农业科学院农业质量标准与检测技术研究所 | A kind of on-line automatic pretreating device of aflatoxin based on flow path switching |
| CN112362607A (en) * | 2018-11-29 | 2021-02-12 | 平顶山市畜产品质量安全监测中心 | Detection cabinet for aflatoxin in corn feed stored in feedlot |
| CN112362607B (en) * | 2018-11-29 | 2023-07-25 | 平顶山市畜产品质量安全监测中心 | Aflatoxin detection cabinet in corn feed stored in feedlot |
| CN110095549A (en) * | 2019-06-12 | 2019-08-06 | 吉林省产品质量监督检验院(吉林省农产品认证中心) | The detection device and method of aflatoxin in a kind of Grain and its product |
| CN110749691A (en) * | 2019-12-23 | 2020-02-04 | 山东畜牧兽医职业学院 | HPLC-MS/MS method for determining aflatoxin and homologue thereof in infant auxiliary food |
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