CN107153101A - A kind of detection method of plant-derived feed polyoxin - Google Patents
A kind of detection method of plant-derived feed polyoxin Download PDFInfo
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- CN107153101A CN107153101A CN201710272176.6A CN201710272176A CN107153101A CN 107153101 A CN107153101 A CN 107153101A CN 201710272176 A CN201710272176 A CN 201710272176A CN 107153101 A CN107153101 A CN 107153101A
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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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The present invention relates to a kind of detection method of plant-derived feed polyoxin, including：The pre-treatment of material；Extract and purify；Qualitative analysis is determined；Quantitative determination；As a result checking is calculated.The present invention has the advantages that identification accuracy height, accurate positioning, representative in the detection of China's polyoxin, has a good application prospect.
Description
Technical field
The invention belongs to Detecting Pesticide field, more particularly to a kind of detection side of plant-derived feed polyoxin
Method.
Background technology
In recent years, with high poison high residue chemical pesticide disabling, the biological pesticide of environmental protection low toxicity and low residue gets over
Favored to get over by peasant.Wherein polyoxin, which has, suppresses the effect that germ production spore and scab expand, and is widely used in preventing
Control cucumber downy mildew, wheat powdery mildew, smut of maize, black fleck disease of ginseng, apple and pears graywall and rice sheath blight disease etc.
Multiple diseases.But polyoxin relatively lags behind to the toxicity research of people and animals at present, toxicological effect situation is still not clear.And it is many
The crops such as paddy rice, corn and the wheat of antimycin pollution are edible by pig, ox and sheep etc. as feed, can accumulate in vivo, pass through
Food chain enters into the human body, can cause certain harm.Current polyoxin assay method has microbial method, capillary electricity
Swimming-electrogenerated chemiluminescence method and high performance liquid chromatography, it is domestic at present using it is more be Capillary Electrophoresis-electrogenerated chemiluminescence
Method and high performance liquid chromatography.And these assay methods are a series of polyoxin metabolins for determining fermentation generation,
Sensitivity is low, and time-consuming, and precision is poor.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of detection method of plant-derived feed polyoxin, the party
Method by using material pre-treatment, extract and purification, qualitative analysis determine, quantitative determination and result calculate checking etc. step pair
Whether detected that there is identification accuracy height, accurate positioning in plant source feed containing polyoxin, it is many in China
It is representative in the detection of antimycin, have a good application prospect.
A kind of detection method of plant-derived feed polyoxin of the present invention, including：
(1) plant-derived Feed Sample is taken, grinds, sieve；Then (mass volume ratio 2.5g is pressed:20ml) add formic acid second
Nitrile solution, is mixed, ultrasonic wave extraction, centrifugation, drying, then is dissolved with ammonia spirit, obtains sample to be clean；
(2) above-mentioned sample to be clean is transferred in extraction column, discarded successively with after acetonitrile, water in advance elution by extraction column
Sample liquid, is eluted with glacial acetic acid acetonitrile solution, collects eluent, drying, constant volume；
(3) qualitative analysis and quantitative determination are carried out using liquid chromatography-tandem mass spectrometry, determined finally by calculating in sample
The content of polyoxin.
The concentration of formic acid acetonitrile solution in the step (1) is 3% (formic acid：Acetonitrile=3:97) -8% (formic acid：Acetonitrile
=8:92)；The concentration of ammonia spirit is 1% (ammoniacal liquor：Water=1:99) -3% (ammoniacal liquor：Water=3:97).
Centrifugal speed in the step (1) is 5000r/min, and centrifugation time is 3-7min.
The concentration of glacial acetic acid acetonitrile solution in the step (2) is 5% (glacial acetic acid：Acetonitrile=5:95) -9% (ice second
Acid：Acetonitrile=9:91).
Extraction column in the step (2) is MAX solid-phase extraction columns.
In the step (3) during qualitative analysis in sample test substance retention time and standard liquid retention time deviation
No more than ± the 3% of standard liquid retention time.
Quantitative determination draws standard curve using extraction standard working solution in the step (3), and ensures test sample
The response of middle polyoxin is in the range of linearity of instrument.
Calculation formula in the step (3) is as follows：
In formula：C：The concentration of corresponding polyoxin in sample, unit is μ g/mL；V：Constant volume, unit is mL；f：
Extension rate；m：Sample mass, unit is g.
The absolute difference of the measurement result independent twice obtained under repeat condition must not exceed the 15% of arithmetic mean of instantaneous value.
Beneficial effect
The present invention is determined by using the pre-treatment of material, extraction and purification, qualitative analysis, quantitative determination and result are calculated
Whether the steps such as checking are high, accurate positioning with identification accuracy to being detected in plant source feed containing polyoxin
Advantage, it is representative in the detection of China's polyoxin, have a good application prospect.
Brief description of the drawings
Fig. 1 is 0.1mg/L polyoxin chromatograms.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
Take the plant-derived feed in the ground such as Hebei, Shandong, Fujian：
(1) pre-treatment of material；Representative Feed Sample 500g, quartering reduction at least 100g are taken, is ground, mistake
0.9mm aperture samples are sieved, and mixing is fitted into closed container, and lucifuge Cord blood is standby；
(2) extract and purify
Extract：2.5g samples are weighed, are inserted in 50.0mL centrifuge tubes, the formic acid acetonitrile solutions of 20mL 4% are added, are vortexed mixed
Even 1min, is placed in ultrasonic wave extraction 30min, and then 5000r/min centrifuges 5min, takes 8mL supernatants, nitrogen instrument blows at 50 DEG C
To near dry, the sample extracted and the 30s that is vortexed are dissolved with 1mL 2.5% ammonia spirit, it is to be clean；
Purification：By mixed-cation solid-phase extraction column successively with after 3.0mL acetonitriles, water in advance elution, by above-mentioned sample to be clean
Product are transferred in post, discard sample solution, are eluted with the glacial acetic acid acetonitrile solutions of 10mL 6%, collect eluent in 20mL teat glasses
In, then with the drying of nitrogen instrument, it is to be measured with 1mL acetonitrile solution constant volumes.
(3) qualitative analysis is determined：Using liquid chromatography tandom mass spectrometry determination：
Chromatogram and Mass Spectrometry Conditions：
Chromatographic column：Hypersil GOLD Amino posts, 30mm × 3.0mm (i.d.), 1.9 μm or suitable specification chromatographic column；
Mobile phase and condition of gradient elution are shown in Table 1：
The mobile phase of table 1 and condition of gradient elution
| Time/min | Flow velocity/(mL/min) | Mobile phase A (1% formic acid)/% | Mobile phase B (methanol)/% |
| 0 | 0.25 | 15 | 85 |
| 0.5 | 0.25 | 15 | 85 |
| 1.0 | 0.25 | 50 | 50 |
| 4.0 | 0.25 | 50 | 50 |
| 5.0 | 0.25 | 15 | 85 |
| 6.0 | 0.25 | 15 | 85 |
Column temperature：40℃；
Sampling volume：5μL；
Ion gun：ESI；
Scan mode：Anion is scanned；
Detection mode：Multiple-reaction monitoring；
Electron spray voltage：4500V；
Atomization gas flow velocity：15.0L/min；
Dry gas stream speed：3.0L/min；
Heating block temperature：400℃；
DL pipe temperature：250℃.
Ionization pattern, qualitative ion pair, quota ion pair, retention time and collision energy reference value are shown in Table 2：
The ionization of table 2 pattern, qualitative ion pair, quota ion pair, retention time and collision energy reference value
Under same experimental conditions, the deviation of test substance retention time and standard liquid retention time is no more than in sample
The relative abundance of the qualitative ion of each component and the close mark of mass concentration in ± 3%, and sample of standard liquid retention time
The relative abundance of corresponding qualitative ion is compared in quasi- solution, and deviation is no more than scope as defined in table 3, then can determine that sample
In there is corresponding determinand.
With respect to the maximum allowable offset (%) of abundance of ions during 3 qualitative confirmation of table
| Relative ion abundance | ＞ 50 | 20~50 | 10~20 | <10 |
| The relative deviation of permission | ±20 | ±25 | ±30 | ±50 |
(4) quantitative determine
Liquid chromatography-tandem mass spectrometry is using external standard-calibration curve method quantitative determination in this standard.To reduce matrix to quantitative
The influence of measure, it is quantitative standard curve to be drawn using extraction standard working solution with standard liquid, and ensure in test sample
The response of polyoxin is in the range of linearity of instrument.Under above-mentioned chromatogram and Mass Spectrometry Conditions, the standard liquid of polyoxin
Multiple-reaction monitoring collection of illustrative plates is shown in Fig. 1.
(5) result calculates checking
The content (X) of polyoxin represents (mg/kg) with mass fraction in sample, is calculated with formula below：
In formula：C：The concentration of corresponding polyoxin B in test liquid, unit is micrograms per millilitre (μ g/mL)；V：Constant volume body
Product, unit is milliliter (mL)；f：Extension rate；m：Sample mass, unit is gram (g).
The absolute difference of the measurement result independent twice obtained under repeat condition must not exceed the 15% of arithmetic mean of instantaneous value.
Detection through above-mentioned steps (1)~(5), it is determined that polyoxin content in detected plant source feed.
As a result it is as follows：
Claims (9)
1. a kind of detection method of plant-derived feed polyoxin, including：
(1) plant-derived Feed Sample is taken, grinds, sieve；Then add formic acid acetonitrile solution, mix, ultrasonic wave extraction, from
The heart, drying, then dissolved with ammonia spirit, obtain sample to be clean；
(2) above-mentioned sample to be clean is transferred in extraction column successively with after acetonitrile, water in advance elution, discards sample solution by extraction column,
Eluted with glacial acetic acid acetonitrile solution, collect eluent, drying, constant volume；
(3) qualitative analysis and quantitative determination are carried out using liquid chromatography-tandem mass spectrometry, determines to resist in sample finally by calculating more
The content of mycin.
2. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the concentration of the formic acid acetonitrile solution in (1) is 3%-8%；The concentration of ammonia spirit is 1%-3%.
3. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the centrifugal speed in (1) is 5000r/min, and centrifugation time is 5-6min.
4. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the concentration of the glacial acetic acid acetonitrile solution in (2) is 5%-9%.
5. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the extraction column in (2) is MAX solid-phase extraction columns.
6. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the deviation of test substance retention time and standard liquid retention time is no more than standard liquid in sample during qualitative analysis in (3)
± the 3% of retention time.
7. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly quantitative determination draws standard curve using extraction standard working solution in (3), and ensures the sound of polyoxin in test sample
It should be worth in the range of linearity of instrument.
8. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：The step
Suddenly the calculation formula in (3) is as follows：
In formula：C：The concentration of corresponding polyoxin in sample, unit is μ g/mL；V：Constant volume, unit is mL；f：Dilution
Multiple；m：Sample mass, unit is g.
9. a kind of detection method of plant-derived feed polyoxin according to claim 1, it is characterised in that：Repeating
Under the conditions of the absolute difference of measurement result independent twice that obtains must not exceed the 15% of arithmetic mean of instantaneous value.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001328A (en) * | 2018-08-17 | 2018-12-14 | 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 LC-MS/MS detection method of polyoxin B |
| CN111307965A (en) * | 2019-12-16 | 2020-06-19 | 陕西麦可罗生物科技有限公司 | Method for detecting polyoxin M by high performance liquid chromatography |
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| CN102914607A (en) * | 2012-11-12 | 2013-02-06 | 谱尼测试科技(天津)有限公司 | High performance liquid chromatography method for measuring residual amount of Fenazaquin in plant-derived food |
| CN103529153A (en) * | 2013-07-26 | 2014-01-22 | 宁波检验检疫科学技术研究院 | Common rapid detection method for various pesticide residues in soybeans |
| CN106248837A (en) * | 2016-10-08 | 2016-12-21 | 江苏省农业科学院 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of fumonisin in a kind of corn |
| CN106546672A (en) * | 2016-10-18 | 2017-03-29 | 吉林农业大学 | A kind of residue analysis method of polyoxin on ginseng |
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Patent Citations (4)
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| CN102914607A (en) * | 2012-11-12 | 2013-02-06 | 谱尼测试科技(天津)有限公司 | High performance liquid chromatography method for measuring residual amount of Fenazaquin in plant-derived food |
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| CN106248837A (en) * | 2016-10-08 | 2016-12-21 | 江苏省农业科学院 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of fumonisin in a kind of corn |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001328A (en) * | 2018-08-17 | 2018-12-14 | 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 LC-MS/MS detection method of polyoxin B |
| CN111307965A (en) * | 2019-12-16 | 2020-06-19 | 陕西麦可罗生物科技有限公司 | Method for detecting polyoxin M by high performance liquid chromatography |
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