CN112067710A - Method for detecting various biotoxins in grain and oil crops - Google Patents
Method for detecting various biotoxins in grain and oil crops Download PDFInfo
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- CN112067710A CN112067710A CN202010803644.XA CN202010803644A CN112067710A CN 112067710 A CN112067710 A CN 112067710A CN 202010803644 A CN202010803644 A CN 202010803644A CN 112067710 A CN112067710 A CN 112067710A
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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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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The invention discloses a method for detecting various biotoxins in grain and oil crops, which is characterized by comprising the following steps: (1) weighing different types of mycotoxin standard samples respectively, dissolving each standard sample by adopting acetonitrile as a solvent, performing constant volume to obtain a mother solution of a standard solution of 1mg/mL, and diluting the mother solution into the standard solution for later use; (2) putting 40g of an original form sample of a to-be-detected sample and 60mL of pure water into a beaker with the volume of 100mL, putting the beaker into an ultrasonic biotoxin extraction device for extraction for 2min, transferring all lower organic phases in the beaker into a centrifuge tube, blowing the organic phases in a water bath at the temperature of below 40 ℃ until the organic phases are dry, adding 2m of acetonitrile and an aqueous solution to dissolve and uniformly mix residues in the centrifuge tube, and filtering by using a filter membrane to obtain a to-be-detected liquid; (3) preparing the obtained standard gradient solution and the solution to be detected, detecting by liquid chromatography-mass spectrometry, recording data, and (4) calculating to respectively obtain the contents of different types of biotoxins in the solution to be detected. The method has accurate result of determining the biotoxin, and provides reliable guarantee for the detection of the mycotoxin in the grains and the pollution risk assessment.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting various biotoxins in grain and oil crops.
Background
The grain and oil raw materials mainly comprise wheat, corn, sorghum, rice and glutinous rice, and contain rich nutrient substances, so that mycotoxin is produced due to the pollution of various fungi in fields and storage processes. For example, fusarium produces Deoxynivalenol (DON), Zearalenone (ZEN) and Fumonisins (FBs), and aspergillus flavus produces Aflatoxins (AFs). The European Union has established the regulation limit and the recommended limit of mycotoxins such as DON, ZEN, AFs, FBs, Ochratoxin (OTA), T-2, HT-2 and the like in corn, and national food safety standards of China stipulate that the DON limit in corn raw grains is 1000 mug/kg, the ZEN limit is 60 mug/kg, the aflatoxin B1(AFB1) limit is 20 mug/kg, and the OTA limit is 5 mug/kg.
The detection method of mycotoxin mainly comprises an enzyme-linked immunosorbent assay, a high performance liquid chromatography, a liquid chromatography-mass spectrometry combined method and the like. The enzyme-linked immunosorbent assay is widely used as a rapid screening method, has the advantages of simple pretreatment and capability of detecting samples in batches, can only detect one mycotoxin at a time, is easily interfered by a grease matrix, generates false positive, and reduces the accuracy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting various biotoxins in grain and oil crops so as to improve the accuracy of mycotoxin detection.
In order to achieve the purpose, the invention provides a method for detecting various biotoxins in grain and oil crops, which comprises the following steps:
(1) preparation of a standard solution: respectively weighing different types of mycotoxin standard samples, respectively dissolving each standard sample by adopting acetonitrile as a solvent, carrying out constant volume to obtain a mother solution of a 1mg/mL standard solution, and diluting the mother solution into the standard solution with a set concentration by using acetonitrile + water solution as required for later use;
(2) preparing a solution to be detected: taking 40g of an original form sample of a detected sample and 60mL of pure water, simultaneously putting the original form sample and the 60mL of pure water into a beaker with the volume of 100mL, putting the beaker into an ultrasonic biotoxin extraction device for extraction for 2min, completely transferring the lower organic phase in the beaker into a centrifuge tube, blowing water bath nitrogen below 40 ℃ to dry, adding 2mL of acetonitrile + aqueous solution to completely dissolve and uniformly mix residues in the centrifuge tube, and filtering through a 0.45-micrometer filter membrane to obtain a liquid to be detected;
(3) respectively taking the prepared standard gradient solution and the solution to be detected, detecting by liquid chromatography-mass spectrometry, recording data,
(4) and (3) calculating the result: and (3) utilizing a liquid chromatography-mass spectrometer with software, quantifying peak areas by an external standard method, and calculating to respectively obtain the content of the untouched biotoxins in the liquid to be detected.
Preferably, the ultrasonic biotoxin extraction device is a 21kHz fixed frequency ultrasonic extraction device.
Preferably, in the step (3), the detected chromatographic-mass spectrometry conditions are as follows:
chromatographic conditions are as follows: mobile phase: 5mmol/L aqueous ammonium acetate solution (A) and acetonitrile (B), flow rate: 0.25ml/min, sample size: 20 μ L, column: inertsil C8(5 μm, 2.1 ID. times.150 mm), column temperature: 30 ℃, column equilibration time: the time for the reaction is 2min,
the gradient elution conditions were as follows: 0-1 min, maintaining the percentage of the acetonitrile phase solvent at 10% for 1min, 1-3 min, and linearly increasing to 60%; 3-5 min, increasing the percentage of the acetonitrile phase solvent from 60% to 95%; 5-10 min, and keeping the percentage of the acetonitrile phase solvent at 95% for 5 min; 10-15 min, and keeping the percentage of the acetonitrile phase solvent at 10% for 5 min;
mass spectrum conditions: an ion source: ESI source, ionization mode: positive and negative ion switching, scan mode: and (5) monitoring multiple reactions.
Preferably, in the step (1), the wine sample is taken by 5ml, the extraction reagent is 10ml of chloroform, the shaking extraction time is 2min, the acetonitrile water solution for redissolution is an acetonitrile + water (3+2) mixed solution, and the adding amount is 1 ml.
Preferably, in the step (1), the mother liquor is diluted by acetonitrile + water solution to be standard solutions with the concentrations of 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00, 10.00, 20.00, 50.00 and 100.00 mu g/L respectively.
Preferably, the mycotoxin standard is selected from the group consisting of aflatoxins, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, zearalenone, ochratoxin A, T-2, HT-2, aflatoxins, fumonisins B1, fumonisins B2, and fumonisins B3.
Preferably, the sample is wheat, corn, sorghum, rice or glutinous rice.
The invention has the beneficial effects that:
the method for detecting various biotoxins in grain and oil crops is simple and convenient to operate, and the adopted instruments are reasonable in price and low in detection cost. The result of the biotoxin determination is accurate and reliable, and reliable technical guarantee is provided for the detection of mycotoxin in grains and products thereof and the pollution risk assessment work.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Examples
(1) Preparation of a standard solution: respectively called from fumonisin B1, fumonisin B2, fumonisin B3, aflatoxinin B1, aflatoxinin B2, aflatoxinin G1, aflatoxinin G2, aflatoxin M1, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, zearalenone, ochratoxin A, T-2, HT-2 and variegated aspergillus toxin 10mg, respectively dissolving each standard sample by adopting acetonitrile as a solvent and fixing the volume to obtain a mother solution of a standard solution, wherein the concentration of the mother solution is 1mg/mL, then diluting the mother solution into the standard solution with a certain concentration by utilizing acetonitrile (3+2) solution as required, and storing at-18 ℃ for later use.
(2) Preparing a solution to be detected: and (3) taking 40g of an original form sample of the sample to be detected and 60mL of pure water, simultaneously putting the sample and the pure water into a beaker with the volume of 100mL, putting the beaker into an ultrasonic biotoxin extraction device for extraction for 2min, transferring all lower organic phases in the beaker into a centrifuge tube, blowing the organic phases in a water bath below 40 ℃ until the organic phases are dried, adding 2mL of acetonitrile and aqueous solution to completely dissolve and uniformly mix residues in the centrifuge tube, and filtering the mixture through a 0.45-micrometer filter membrane to obtain the liquid to be detected.
(3) And (3) liquid chromatography-mass spectrometry detection:
chromatographic conditions
Mobile phase: 5mmol/L ammonium acetate aqueous solution (A) and acetonitrile (B);
flow rate: 0.25 ml/min;
sample introduction amount: 20 mu L of the solution;
a chromatographic column: InertsilC8(5 μm, 2.1 ID. times.150 mm);
column temperature: 30 ℃;
chromatographic column equilibration time: 2 min;
the gradient elution conditions were as follows: 0-1 min, maintaining the percentage of the acetonitrile phase solvent at 10% for 1min, 1-3 min, and linearly increasing to 60%; 3-5 min, increasing the percentage of the acetonitrile phase solvent from 60% to 95%; 5-10 min, and keeping the percentage of the acetonitrile phase solvent at 95% for 5 min; 10-15 min, and keeping the percentage of the acetonitrile phase solvent at 10% for 5 min.
Conditions of Mass Spectrometry
An ion source: an ESI source;
an ionization mode: switching positive ions and negative ions;
scanning mode: and (5) monitoring multiple reactions.
(4) And (3) determination: respectively taking the prepared standard gradient solution, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00, 10.00, 20.00, 50.00 and 100.00 mu g/L standard solution and solution to be detected, carrying out sample injection detection by adopting the chromatographic mass spectrometry conditions, recording data and obtaining a linear regression equation of 16 standard samples; and quantitatively calculating through workstation software to obtain the residual quantity of the toxin in the sample in the liquid to be detected.
The following sensitivity tests were performed on the method of the invention: the sensitivity test comprises the sensitivity of an instrument and the sensitivity of a method, the sensitivity of the instrument is expressed by the detection limit of the instrument, and the minimum concentration of the biotoxin mixed standard solution with the signal-to-noise ratio of more than or equal to 3 is taken as the detection limit of the instrument; the sensitivity of the method is expressed by the quantitative limit of the method, and the minimum concentration of the biotoxin mixed standard solution with the signal-to-noise ratio of more than or equal to 10 is taken as the quantitative limit of the method. The relevant data obtained are shown in Table 1.
The following accuracy and reproducibility experiments were performed on the method of the present invention: selecting the same grain sample as a blank sample after pretreatment, dividing the blank sample into 3 parts, respectively adding mixed standard working solution to perform a standard addition recovery experiment, and calculating the recovery rate; selecting 1 grain sample to process 6 grain samples according to the same pretreatment method, respectively carrying out experiments, and judging the reproducibility of the analysis method by calculating the range of Relative Standard Deviation (RSD). The recovery rate is 90-120%, and the RSD is less than 10%.
The above examples demonstrate that the method of the present invention can accurately perform qualitative and quantitative detection on biotoxin in grain and oil raw materials.
. Therefore, the scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the scope of the present invention, which is defined by the claims of the present patent application.
Claims (7)
1. A method for detecting a plurality of biotoxins in grain and oil crops is characterized by comprising the following steps:
(1) preparation of a standard solution: respectively weighing different types of mycotoxin standard samples, respectively dissolving each standard sample by adopting acetonitrile as a solvent, carrying out constant volume to obtain a mother solution of a 1mg/mL standard solution, and diluting the mother solution into the standard solution with a set concentration by using acetonitrile + water solution as required for later use;
(2) preparing a solution to be detected: taking 40g of an original form sample of a detected sample and 60mL of pure water, simultaneously putting the original form sample and the 60mL of pure water into a beaker with the volume of 100mL, putting the beaker into an ultrasonic biotoxin extraction device for extraction for 2min, completely transferring the lower organic phase in the beaker into a centrifuge tube, blowing water bath nitrogen below 40 ℃ to dry, adding 2m of acetonitrile + aqueous solution to completely dissolve and uniformly mix residues in the centrifuge tube, and filtering through a 0.45-micrometer filter membrane to obtain a liquid to be detected;
(3) respectively preparing the obtained standard gradient solution and the solution to be detected, detecting by liquid chromatography-mass spectrometry, and recording data;
(4) and (3) calculating the result: and (3) utilizing the self-contained software of the LC-MS instrument and the peak area quantification of an external standard method to calculate and respectively obtain the contents of different biotoxins in the liquid to be detected.
2. The method of claim 1, wherein the ultrasonic biotoxin extraction device is a 21kHz fixed frequency ultrasonic extraction device.
3. The method of claim 2, wherein in step (3), the detected chromatography-mass spectrometry conditions are:
chromatographic conditions are as follows: mobile phase: 5mmo L/L ammonium acetate aqueous solution (A) and acetonitrile (B), flow rate: 0.25ml/min, sample size: 20 μ L, column: inertsil C8(5 μm, 2.1 ID. times.150 mm), column temperature: 30 ℃, column equilibration time: the time for the reaction is 2min,
the gradient elution conditions were as follows: 0-1 min, maintaining the percentage of the acetonitrile phase solvent at 10% for 1min, 1-3 min, and linearly increasing to 60%; 3-5 min, increasing the percentage of the acetonitrile phase solvent from 60% to 95%; 5-10 min, and keeping the percentage of the acetonitrile phase solvent at 95% for 5 min; 10-15 min, and keeping the percentage of the acetonitrile phase solvent at 10% for 5 min;
mass spectrum conditions: an ion source: ESI source, ionization mode: positive and negative ion switching, scan mode: and (5) monitoring multiple reactions.
4. The method according to claim 1, wherein in the step (1), the amount of the wine sample is 5ml, the extraction reagent is 10ml of chloroform, the extraction time is 2min under shaking, the aqueous acetonitrile solution for redissolution is a mixed solution of acetonitrile and water (3+2), and the amount of the mixture is 1 ml.
5. The method according to claim 1, wherein in the step (1), the mother liquor is diluted with acetonitrile + water solution into standard solutions with the concentrations of 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00, 10.00, 20.00, 50.00 and 100.00 μ g/L.
6. The method of claim 1, wherein the mycotoxin standard is selected from the group consisting of fumonisin B1, fumonisin B2, fumonisin B3, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, zearalenone, ochratoxin A, T-2, HT-2, and aflatoxins.
7. The method of claim 1, wherein the sample is wheat, corn, sorghum, rice or glutinous rice.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113390992A (en) * | 2021-06-15 | 2021-09-14 | 浙江海正药业股份有限公司 | Method for analyzing and detecting trace aflatoxin in water-soluble fermented medicine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113390992A (en) * | 2021-06-15 | 2021-09-14 | 浙江海正药业股份有限公司 | Method for analyzing and detecting trace aflatoxin in water-soluble fermented medicine |
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Application publication date: 20201211 |