CN111122737A - Detection method for mycotoxin pollution in agricultural products - Google Patents
Detection method for mycotoxin pollution in agricultural products Download PDFInfo
- Publication number
- CN111122737A CN111122737A CN201911391365.0A CN201911391365A CN111122737A CN 111122737 A CN111122737 A CN 111122737A CN 201911391365 A CN201911391365 A CN 201911391365A CN 111122737 A CN111122737 A CN 111122737A
- Authority
- CN
- China
- Prior art keywords
- desorption
- sample
- mycotoxin
- liquid
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
-
- 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/95—Detectors specially adapted therefor; Signal analysis
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention discloses a method for detecting mycotoxin pollution in agricultural products, which comprises the following steps: preparing an adsorption phase, leaching, desorbing and detecting; the method adopts aluminosilicate and active carbon as raw materials to prepare the adsorption phase, reduces the condition that the detection result has deviation due to less extracted mycotoxin content or other impurities mixed in the sample when the mycotoxin content of an agricultural product sample is detected by adopting a mode of leaching, adsorbing and desorbing, and improves the detection precision.
Description
Technical Field
The present invention relates to the field of mycotoxin detection. More particularly, the present invention relates to a method for detecting mycotoxin contamination in agricultural products.
Background
Mycotoxins are secondary metabolites produced by fungi, are widely present in agricultural products such as grain and oil, can produce fatal results for both human beings and animals, are not in one-to-one correspondence with the types of the toxins, can cause the occurrence of a situation that the fungi can produce a plurality of toxins, has additive effects on the toxicity of the mycotoxins, and has large variation in toxicity depending on the types/amount/intake amount of the toxins, the continuous intake time, environmental factors and the like. The mycotoxins with great harm mainly comprise aflatoxin, zearalenone, vomitoxin, ochratoxin A, fumonisin, T2/HT2 toxin and the like.
The detection of mycotoxins in agricultural products has become critical for the reasons mentioned above; the existing detection methods are various, but the basic operations are sample crushing, extraction, filtration, purification and sample injection analysis. The extraction of mycotoxin in a sample has a great influence on subsequent detection, the inaccurate polarity of solvent selection can cause less extraction amount or more protein or starch impurities of agricultural products, and both have great influence on the detection result, so an effective extraction and detection method for mycotoxin is needed.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for detecting mycotoxin contamination in agricultural products, which adopts aluminosilicate and active carbon as raw materials to prepare an adsorption phase, adopts a mode of leaching, adsorbing and desorbing firstly to reduce the condition that the detection result is deviated due to less content of extracted mycotoxin or other impurities mixed in the sample when the content of mycotoxin is detected in an agricultural product sample, improves the detection precision, adopts a clamping mode to the sample and the adsorption phase, enables the adsorption phase to be reused and the sample to be recycled compared with the prior operation of directly mixing the sample with the adsorption phase, adopts a desorption device to carry out desorption operation, utilizes the pressure of evaporation expansion of desorption liquid to carry out pressure desorption on the mycotoxin adsorbed on the adsorption phase, and enables the desorption effect to be more complete compared with the prior normal-pressure heating soaking mode, and the consumption of desorption liquid is less, and the subsequent detection precision is indirectly improved.
To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a method for detecting mycotoxin contamination in a live agricultural product, comprising the steps of:
preparing an adsorption phase, namely preparing one or more of iron pyrite, activated carbon, artificial zeolite or diatomite into the adsorption phase, pretreating the adsorption phase by using a phosphate buffer solution with the pH value of 6-8, pressing the obtained adsorption phase into tablets with the thickness of 2-5 cm, and wrapping the tablets by using microfiber filter cloth to obtain a finished product of the adsorption phase; weighing and crushing a sample of an agricultural product to be detected, coating the sample by using microfiber filter cloth, and clamping the adsorption phase and the sample to obtain an extraction sample;
step two, leaching the leaching liquor for the extraction sample for more than three times, soaking for 10-15 min each time, carrying out thin-layer chromatography detection and confirmation on the leaching liquor obtained after leaching by adopting a bidirectional expansion mode at the end point of leaching times until no mycotoxin is detected in the leaching liquor, and combining eluents obtained after leaching liquor is repeatedly extracted to obtain a total extracting solution and an extracted sample; wherein the leaching liquor is prepared from acetonitrile and water according to the volume ratio of 8: 2, and the mass of the eluent is 5-10 times that of the sample;
removing the sample between the extracted samples, reserving the adsorption phase, and performing desorption operation by using the heated desorption solution to obtain a total desorption solution; the desorption solution is methanol, acetic acid and water according to the weight ratio of 95: 4: 1, performing thin-layer chromatography detection on desorption liquid obtained after desorption in a bidirectional unfolding mode, and confirming a desorption end point;
combining the total extracting solution and the total desorption solution, performing nitrogen blowing concentration at the temperature of 40-45 ℃ in vacuum, and adding acetonitrile water solution with the mass fraction of 50% to a constant volume to obtain a sample to be detected;
preparing a standard solution of vomitoxin, aflatoxin, ochratoxin, T-2 toxin, fumonisin, zearalenone, fusarenol, variegated triptycetin and patulin by adopting an acetonitrile water solution with the mass fraction of 50%, detecting the standard solution by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, and drawing a standard curve;
and step four, detecting the mycotoxin in the sample to be detected by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, obtaining the corresponding concentration of the mycotoxin in the detection solution by contrasting a standard curve, and calculating the content of the mycotoxin in the sample.
Preferably, the activated carbon or the artificial zeolite is soaked by benzyldimethyloctadecyl ammonium chloride hydrate before use.
Preferably, the chromatographic conditions are: the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column, 4.6X 150mm, 3.5 μm; the column temperature is 30 ℃; the sample volume is 10 mu L; mobile phase: a is 0.03 percent formic acid and 1mM ammonium acetate aqueous solution; b is methanol; gradient elution: 0-8 min.22% -36% of B; 8-16 min, 36-72% of B; 16-27 min, 72-93% B; rinsing with 93% B for 10 min; before each sample injection, the column was equilibrated with 20% B for 5 min;
the mass spectrum conditions are as follows: the ion source is an electrospray ion source; the ionization mode is ESI +; the detection mode is multi-reaction detection, and the capillary voltage is 3.0 kV; atomizer pressure was 40 psi; the drying airflow rate is 10L/min; the drying gas temperature was 300 ℃.
Preferably, the desorption operation in the second step is performed in a desorption apparatus comprising:
the upper part of the desorption chamber is provided with an opening, the edge of the outer wall of the opening is provided with a clamping sealing ring, a heating pipe is arranged in the desorption chamber, and the heating pipe is connected with a power supply;
the filling funnel is in a Buchner funnel shape, the outer diameter of the filling funnel is slightly smaller than the diameter of the upper opening of the desorption chamber, the upper edge of the filling funnel is turned outwards to be erected in the desorption chamber, and the filling layer of the filling funnel is higher than the liquid level of desorption liquid in the desorption chamber;
the collecting chamber is provided with a sealing cover at the upper part of the desorption chamber, the bottom surface of the collecting chamber is raised upwards, the end part of the collecting chamber is opened, a liquid passing channel communicated with the desorption chamber is formed, the height of the liquid passing channel is more than half of the height of the collecting chamber, the upper part of the collecting chamber is opened, and the upper part of the collecting chamber is provided with a threaded sealing cover; the end part of the liquid outlet channel is provided with a liquid outlet valve seat, the lower end of the liquid outlet valve seat is fixedly connected with the liquid outlet channel through threads, a valve cover is movably sleeved on the liquid outlet valve seat, the lower end of the valve cover is movably abutted against the lower end of the liquid outlet valve seat, and the lower bottom surface of the valve cover is provided with a plurality of liquid outlet holes;
wherein, the lowest department of collecting the room is equipped with the desorption liquid export that runs through, the lower part of desorption room is equipped with the desorption liquid import that runs through.
Preferably, a sealing ring is arranged at the joint of the liquid outlet valve seat and the liquid feeding channel, and a sealing gasket is arranged at the joint of the desorption chamber and the collection chamber.
Preferably, the desorption liquid inlet is provided with a one-way valve.
The invention at least comprises the following beneficial effects:
firstly, aluminosilicate, activated carbon and the like are used as raw materials to prepare an adsorption phase, so that the condition that the detection result is deviated due to the fact that the content of extracted mycotoxin is low or other impurities mixed in a sample are reduced when the content of mycotoxin in an agricultural product sample is detected, and the detection precision is improved; the desorption liquid is heated to the micro-boiling degree, the adsorption phase and desorption liquid action area is greatly increased, the desorption effect is improved, the concentration of the mycotoxin in the obtained desorption liquid is higher, the detection precision is improved, meanwhile, the higher recovery rate of the detected mycotoxin is ensured, and the loss generated by extraction operation is reduced;
secondly, the sample and the adsorption phase are clamped, microfiber filter cloth is arranged in the middle of the sample and the adsorption phase at intervals, and purification operation is completed in the leaching process; meanwhile, compared with the existing operation processes of sample crushing, extraction, filtration, purification and sample injection analysis, the microfiber filter cloth arranged in the middle at intervals directly synthesizes the extraction, filtration and purification into one step, thereby simplifying the operation process;
thirdly, a desorption device is adopted for desorption operation, the desorption liquid is enabled to be slightly boiled by heating the desorption liquid, the air pressure in the desorption chamber is slightly larger than the external air pressure under the action of a valve cover, so that the movement of solvent molecules of the desorption liquid is accelerated, the contact with an adsorption phase is more sufficient, the desorption of mycotoxin is facilitated, the desorption effect is better compared with the effect of soaking desorption operation, the desorption time is reduced under the condition of equivalent toxin, the amount of the used desorption liquid is less, and the subsequent detection precision and efficiency are indirectly improved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the structure and liquid running of a desorption device according to one embodiment of the present invention;
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
< example 1>
A method for detecting mycotoxin contamination in agricultural products, the method comprising the steps of:
step one, preparation of an adsorption phase, namely mixing the biogas iron ore and the activated carbon according to the ratio of 1: 5, uniformly mixing and stirring to obtain an adsorption phase, soaking the adsorption phase in a phosphate buffer solution with the pH value of 6-8, pressing the obtained adsorption phase into tablets with the thickness of 2-5 cm, and wrapping the tablets with microfiber filter cloth to obtain a finished adsorption phase product;
preparing a sample, namely weighing and crushing a sample of an agricultural product to be detected, coating the sample by using microfiber filter cloth, clamping the adsorption phase and the sample, wherein the weight of the used adsorption phase is 10 times of that of the sample to be detected, and obtaining an extracted sample, compacting, binding and fixing;
and step two, leaching the leaching liquor for extracting the sample for three times, wherein each time of soaking is 10-15 min, and the leaching liquor is acetonitrile and water according to a volume ratio of 8: 2, the mass of the eluent is 8 times that of the sample, and when the third leaching is carried out, mycotoxin cannot be detected by the thin-layer chromatography, and the thin-layer chromatography detection adopts a bidirectional development mode; mixing eluents obtained after multiple times of leaching to obtain a total extracting solution and a leached extraction sample;
disassembling the extracted samples after leaching, removing samples among the extracted samples, reserving an adsorption phase, performing desorption operation by using heated desorption liquid, specifically putting the adsorption phase into a Buchner funnel, flushing by using the desorption liquid heated to 50-60 ℃, and combining the flushed desorption liquid to obtain total desorption liquid; the desorption solution is methanol, acetic acid and water according to the weight ratio of 95: 4: 1, and the detection method is the same as a thin-layer chromatography detection method in the leaching operation;
combining the total extracting solution and the total desorption solution, performing nitrogen blowing concentration at the temperature of 40-45 ℃ in vacuum, and adding acetonitrile water solution with the mass fraction of 50% to a constant volume to obtain a sample to be detected;
thirdly, preparing a standard solution of aflatoxin B1, ochratoxin, vomitoxin and zearalenone by adopting an acetonitrile water solution with the mass fraction of 50%, detecting the standard solution by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, and drawing a standard curve;
step four, detecting the mycotoxin in the sample to be detected by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, obtaining the corresponding concentration of the mycotoxin in the detection solution by contrasting a standard curve, and calculating the content of the mycotoxin in the sample;
the chromatographic conditions are as follows:
the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column, 4.6X 150mm, 3.5 μm; the column temperature is 30 ℃; the sample volume is 10 mu L;
mobile phase: a is 0.03 percent formic acid and 1mM ammonium acetate aqueous solution; b is methanol;
gradient elution: 0-8 min.22% -36% of B; 8-16 min, 36-72% of B; 16-27 min, 72-93% B; rinsing with 93% B for 10 min; before each sample injection, the column was equilibrated with 20% B for 5 min;
the mass spectrum conditions are as follows: the ion source is an electrospray ion source; the ionization mode is ESI +; the detection mode is multi-reaction detection, and the capillary voltage is 3.0 kV; atomizer pressure was 40 psi; the drying airflow rate is 10L/min; the drying gas temperature was 300 ℃.
< example 2>
A method for detecting mycotoxin contamination in agricultural products, the method comprising the steps of:
step one, preparation of an adsorption phase, namely mixing the iron ore, the artificial zeolite or the diatomite according to a ratio of 1: 5: 2, mixing and stirring uniformly to obtain an adsorption phase; soaking the artificial zeolite by benzyl dimethyl octadecyl ammonium chloride hydrate for 15-30 min before use, wherein the mass of the used benzyl dimethyl octadecyl ammonium chloride hydrate is 1.5 times that of the artificial zeolite; soaking the adsorption phase in phosphate buffer solution with the pH value of 6-8 for 5-10 min; pressing the obtained adsorption phase into a pressing sheet with the thickness of 2-5 cm, and wrapping the pressing sheet with microfiber filter cloth to obtain a finished adsorption phase product;
preparing a sample, namely weighing and crushing a sample of an agricultural product to be detected, coating the sample by using microfiber filter cloth, and clamping the adsorption phase and the sample, wherein the sample can be divided into multiple parts for clamping, and the total weight of the used adsorption phase is 10 times of the total weight of the sample to be detected, so as to obtain an extracted sample, compacting, binding and fixing;
and step two, leaching the leaching liquor for extracting the sample for three times, wherein each time of soaking is 10-15 min, and the leaching liquor is acetonitrile and water according to a volume ratio of 8: 2, the mass of the eluent is 8 times that of the sample, and when the third leaching is carried out, mycotoxin cannot be detected by the thin-layer chromatography, and the thin-layer chromatography detection adopts a bidirectional development mode; mixing eluents obtained after multiple times of leaching to obtain a total extracting solution and a leached extraction sample;
disassembling the extracted samples after leaching, removing samples among the extracted samples, reserving an adsorption phase, performing desorption operation by using heated desorption liquid, specifically putting the adsorption phase into a Buchner funnel, flushing by using the desorption liquid heated to 50-60 ℃, and combining the flushed desorption liquid to obtain total desorption liquid; the desorption solution is methanol, acetic acid and water according to the weight ratio of 95: 4: 1, and the detection method is the same as a thin-layer chromatography detection method in the leaching operation;
combining the total extracting solution and the total desorption solution, performing nitrogen blowing concentration at the temperature of 40-45 ℃ in vacuum, and adding acetonitrile water solution with the mass fraction of 50% to a constant volume to obtain a sample to be detected;
preparing a series of standard solutions of mycotoxins (vomitoxin, aflatoxin, ochratoxin, T-2 toxin, fumonisin, zearalenone, fusarenol, variolomycin, patulin and the like) by adopting an acetonitrile water solution with the mass fraction of 50%, detecting the standard solutions by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, and drawing a standard curve;
and step four, detecting the mycotoxin in the sample to be detected by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, obtaining the corresponding concentration of the mycotoxin in the detection solution by contrasting a standard curve, and calculating the content of the mycotoxin in the sample.
The chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column, 4.6X 150mm, 3.5 μm; the column temperature is 30 ℃; the sample volume is 10 mu L; mobile phase: a is 0.03 percent formic acid and 1mM ammonium acetate aqueous solution; b is methanol; gradient elution: 0-8 min.22% -36% of B; 8-16 min, 36-72% of B; 16-27 min, 72-93% B; rinsing with 93% B for 10 min; before each sample injection, the column was equilibrated with 20% B for 5 min;
the mass spectrum conditions are as follows: the ion source is an electrospray ion source; the ionization mode is ESI +; the detection mode is multi-reaction detection, and the capillary voltage is 3.0 kV; atomizer pressure was 40 psi; the drying airflow rate is 10L/min; the drying gas temperature was 300 ℃.
< example 3>
A method for detecting mycotoxin contamination in agricultural products, the method comprising the steps of:
step one, preparation of an adsorption phase, namely preparing the biogas iron ore and activated carbon according to the ratio of 1: 5, uniformly mixing and stirring to obtain an adsorption phase, soaking the adsorption phase in a phosphate buffer solution with the pH value of 6-8, pressing the obtained adsorption phase into tablets with the thickness of 2-5 cm, and wrapping the tablets with microfiber filter cloth to obtain a finished adsorption phase product;
preparing a sample, namely weighing and crushing a sample of an agricultural product to be detected, coating the sample by using microfiber filter cloth, clamping the adsorption phase and the sample, wherein the weight of the used adsorption phase is 10 times of that of the sample to be detected, and obtaining an extracted sample, compacting, binding and fixing;
step two, leaching the leaching liquor for the extraction sample for more than three times, soaking for 10-15 min each time, carrying out thin-layer chromatography detection and confirmation on the leaching liquor obtained after leaching by adopting a bidirectional expansion mode at the end point of leaching times until no mycotoxin is detected in the leaching liquor, and combining eluents obtained after leaching liquor is repeatedly extracted to obtain a total extracting solution and an extracted sample; wherein, the eluent is acetonitrile and water according to the volume ratio of 8: 2, and the mass of the eluent is 5-10 times that of the sample;
removing the sample between the extracted samples, reserving the adsorption phase, and performing desorption operation by using the heated desorption solution to obtain a total desorption solution;
wherein the desorption operation is performed in a desorption apparatus, as shown in fig. 1, comprising:
the desorption chamber 1 is provided with an opening at the upper part, the edge of the outer wall of the opening is provided with a clamping-connected sealing ring, a heating pipe is arranged in the desorption chamber 1, and the heating pipe is connected with a power supply;
a filling funnel 4 which is in a Buchner funnel shape, the outer diameter of the filling funnel 4 is slightly smaller than the diameter of the upper opening of the desorption chamber 1, the upper edge of the filling funnel 4 is turned outwards to be erected in the desorption chamber 1, and the filling layer of the filling funnel 4 is higher than the liquid level of desorption liquid in the desorption chamber 1;
a collection chamber 2, wherein a sealing cover 3 of the collection chamber 2 is arranged at the upper part of the desorption chamber 1, the bottom surface of the collection chamber 2 is raised upwards, the end part of the collection chamber is opened, a liquid passing channel 8 communicated with the desorption chamber 1 is formed, the height of the liquid passing channel 8 is more than half of the height of the collection chamber 2, the upper part of the collection chamber 2 is opened, and a thread sealing cover 3 is arranged at the upper part of the collection chamber 2; a liquid outlet valve seat 5 is arranged at the end part of the liquid outlet channel 8, the lower end of the liquid outlet valve seat 5 is fixedly connected with the liquid outlet channel 8 through threads, a valve cover 6 is movably sleeved on the liquid outlet valve seat 5, the lower end of the valve cover 6 is movably abutted with the lower end of the liquid outlet valve seat 5, and a plurality of liquid outlet holes 9 are formed in the lower bottom surface of the valve cover 6;
wherein, the lowest part of the collection chamber 2 is provided with a penetrated desorption liquid outlet, the lower part of the desorption chamber 1 is provided with a penetrated desorption liquid inlet, and the desorption liquid inlet is provided with a one-way valve; a sealing ring is arranged at the joint of the liquid outlet valve seat 5 and the liquid feeding channel, and a sealing gasket 7 is arranged at the joint of the desorption chamber 1 and the collection chamber 2;
the specific use method comprises the steps of introducing desorption liquid into the desorption chamber 1 through a desorption liquid inlet at the lower part, filling the adsorption phase after extraction into the collection chamber 2, finishing the installation of the device, adsorbing most of mycotoxin into the adsorption phase at the moment, electrifying a heating pipe in the desorption chamber 1, heating the desorption liquid, boiling the desorption liquid slightly when the temperature of the desorption liquid rises to be higher than 50 ℃, lifting the desorption liquid along a liquid passage 8, jacking a valve cover 6 by vapor pressure, allowing the desorption liquid with the mycotoxin to enter the collection chamber 2 through a liquid outlet hole 9 below the valve cover after desorption, finishing the desorption operation, detecting the desorption liquid flowing out from a desorption liquid outlet through TLC (thin layer chromatography), and judging the end point of the desorption operation.
Wherein the desorption solution is methanol, acetic acid and water according to the weight ratio of 95: 4: 1, performing thin-layer chromatography detection on desorption liquid obtained after desorption in a bidirectional unfolding mode, and confirming a desorption end point;
combining the total extracting solution and the total desorption solution, performing nitrogen blowing concentration at the temperature of 40-45 ℃ in vacuum, and adding acetonitrile water solution with the mass fraction of 50% to a constant volume to obtain a sample to be detected;
preparing a standard solution of vomitoxin, aflatoxin, ochratoxin, T-2 toxin, fumonisin, zearalenone, fusarenol, variegated triptycetin and patulin by adopting an acetonitrile water solution with the mass fraction of 50%, detecting the standard solution by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, and drawing a standard curve;
and step four, detecting the mycotoxin in the sample to be detected by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, obtaining the corresponding concentration of the mycotoxin in the detection solution by contrasting a standard curve, and calculating the content of the mycotoxin in the sample.
The chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column, 4.6X 150mm, 3.5 μm; the column temperature is 30 ℃; the sample volume is 10 mu L; mobile phase: a is 0.03 percent formic acid and 1mM ammonium acetate aqueous solution; b is methanol; gradient elution: 0-8 min.22% -36% of B; 8-16 min, 36-72% of B; 16-27 min, 72-93% B; rinsing with 93% B for 10 min; before each sample injection, the column was equilibrated with 20% B for 5 min;
the mass spectrum conditions are as follows: the ion source is an electrospray ion source; the ionization mode is ESI +; the detection mode is multi-reaction detection, and the capillary voltage is 3.0 kV; atomizer pressure was 40 psi; the drying airflow rate is 10L/min; the drying gas temperature was 300 ℃.
Wherein, the difference of the embodiment 1 to the embodiment 3 is that the means for extracting the mycotoxin are different, the used adsorption phases are different, and the adopted thin layer chromatography detection method/high performance liquid chromatography and the mass spectrum detection means are the same;
the standard curves of the toxins of various fungi detected by the methodology can be universal;
analyzing the detection results of the detection modes of the embodiments 1 to 3 by adopting a methodology method, and taking wheat flour without mycotoxin as a verification sample;
detection means and indexes:
thin-layer chromatography:
the developing solution used in the TLC detection is different in detection toxin and different in developing agent, and common developing agents include: the aflatoxin is: v (acetone): v (chloroform) ═ 9: 1; the vomitoxin is: v (acetonitrile): v (water) ═ 21: 4; the variolothricin is: v (toluene): v (methanol): v (acetic acid) ═ 90: 8: 2; detecting by using an ultraviolet/fluorescent lamp detector;
high performance liquid chromatography:
standard curve plotting of mycotoxins:
selecting representative mycotoxins (including aflatoxin B1, ochratoxin A, zearalenone and vomitoxin), preparing a standard mixed solution, measuring the detection limit of each toxin by a measuring instrument, adjusting the concentration of the standard solution according to the detection limit, and determining a linear equation, a correlation coefficient and a linear range of the mycotoxins, wherein the prepared concentration is shown in table 1;
TABLE 1
Because the detection means are the same, the standard solution and the instrument in the embodiments 1 to 3 are obtained through detection, the drawn linear equation/correlation coefficient and the linear range can be universal, and the detection is not the detection key point of the invention, so the detection is omitted;
detecting the recovery rate, namely performing 3 levels of addition recovery experiments by taking wheat without toxins as a blank sample matrix, performing 3 determination method quantitative limits (which can be a standard solution 5) of each mycotoxin with the addition amount of 1 time, 2 times and 10 times respectively, performing 6 times of repeated experiments at each concentration level, analyzing and determining the recovery rate and counting;
the above recovery test mainly measures the difference between the extraction means of the three examples;
the reference conditions of mycotoxin mass spectrometry are shown in table 2;
TABLE 2
The invention takes wheat without toxin as a substrate, and the data of the methodological verification of the detection related results of the detection method of the embodiment 1-3 are shown in the table 3;
TABLE 3
The aluminosilicate, the activated carbon and the like are adopted as raw materials to prepare the adsorption phase, so that the condition that the detection result has deviation due to the fact that the content of the extracted mycotoxin is low or other impurities mixed in the sample are mixed when the content of the mycotoxin in an agricultural product sample is detected is reduced, the recovery rate is high, and the relative standard deviation is remarkably reduced; the linear range and detection limit of the three embodiments are the same, since the detection means are the same.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (6)
1. A method for detecting mycotoxin contamination in an agricultural product, comprising the steps of:
preparing an adsorption phase, namely preparing one or more of iron pyrite, activated carbon, artificial zeolite or diatomite into the adsorption phase, pretreating the adsorption phase by using a phosphate buffer solution with the pH value of 6-8, pressing the obtained adsorption phase into tablets with the thickness of 2-5 cm, and wrapping the tablets by using microfiber filter cloth to obtain a finished product of the adsorption phase; weighing and crushing a sample of an agricultural product to be detected, coating the sample by using microfiber filter cloth, and clamping the adsorption phase finished product and the sample to obtain an extraction sample;
step two, leaching the leaching liquor for the extraction sample for more than three times, soaking for 10-15 min each time, carrying out thin-layer chromatography detection and confirmation on the leaching liquor obtained after leaching by adopting a bidirectional expansion mode at the end point of leaching times until no mycotoxin is detected in the leaching liquor, and combining the leaching liquor for multiple times to obtain a total extracting solution and an extracted sample after leaching; wherein the leaching liquor is prepared from acetonitrile and water according to the volume ratio of 8: 2, and the mass of the leaching liquor is 5-10 times that of the sample;
removing the sample between the extracted samples to obtain an adsorption phase finished product adsorbed with the mycotoxin, and desorbing the adsorption phase finished product adsorbed with the mycotoxin by using the heated desorption solution to obtain a total desorption solution; the desorption solution is methanol, acetic acid and water according to the weight ratio of 95: 4: 1, performing thin-layer chromatography detection on desorption liquid obtained after desorption in a bidirectional unfolding mode, and confirming a desorption end point;
combining the total extracting solution and the total desorption solution, performing nitrogen blowing concentration at the temperature of 40-45 ℃ in vacuum, and adding acetonitrile water solution with the mass fraction of 50% to a constant volume to obtain a sample to be detected;
thirdly, preparing various standard solutions of vomitoxin, aflatoxin, ochratoxin, T-2 toxin, fumonisin, zearalenone, fusarenol, variegated aspergillocin and patulin by adopting an acetonitrile water solution with the mass fraction of 50%, detecting the standard solutions by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, and drawing a standard curve;
and step four, detecting the mycotoxin in the sample to be detected by adopting high performance liquid chromatography and triple quadrupole tandem mass spectrometry, obtaining the corresponding concentration of the mycotoxin in the detection solution by contrasting a standard curve, and calculating the content of the mycotoxin in the sample.
2. The method of detecting mycotoxin contamination in an agricultural product of claim 1, wherein the activated carbon or the artificial zeolite is soaked with benzyldimethyloctadecyl ammonium chloride hydrate prior to use.
3. A method of detecting mycotoxin contamination in an agricultural product as defined in claim 1, wherein the chromatographic conditions are: the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 column, 4.6X 150mm, 3.5 μm; the column temperature is 30 ℃; the sample volume is 10 mu L; mobile phase: a is 0.03 percent formic acid and 1mM ammonium acetate aqueous solution; b is methanol; gradient elution: 0-8 min.22% -36% of B; 8-16 min, 36-72% of B; 16-27 min, 72-93% B; rinsing with 93% B for 10 min; before each sample injection, the column was equilibrated with 20% B for 5 min;
the mass spectrum conditions are as follows: the ion source is an electrospray ion source; the ionization mode is ESI +; the detection mode is multi-reaction detection, and the capillary voltage is 3.0 kV; atomizer pressure was 40 psi; the drying airflow rate is 10L/min; the drying gas temperature was 300 ℃.
4. A method of detecting mycotoxin contamination in an agricultural product as defined in claim 1, wherein the desorbing operation of step two is performed in a desorbing device comprising:
the upper part of the desorption chamber is provided with an opening, the edge of the outer wall of the opening is provided with a clamping sealing ring, a heating pipe is arranged in the desorption chamber, and the heating pipe is connected with a power supply;
the filling funnel is in a Buchner funnel shape, the outer diameter of the filling funnel is slightly smaller than the diameter of the upper opening of the desorption chamber, the upper edge of the filling funnel is turned outwards to be erected in the desorption chamber, and the filling layer of the filling funnel is higher than the liquid level of desorption liquid in the desorption chamber;
the collecting chamber is provided with a sealing cover at the upper part of the desorption chamber, the bottom surface of the collecting chamber is raised upwards, the end part of the collecting chamber is opened, a liquid passing channel communicated with the desorption chamber is formed, the height of the liquid passing channel is more than half of the height of the collecting chamber, the upper part of the collecting chamber is opened, and the upper part of the collecting chamber is provided with a threaded sealing cover; the end part of the liquid outlet channel is provided with a liquid outlet valve seat, the lower end of the liquid outlet valve seat is fixedly connected with the liquid outlet channel through threads, a valve cover is movably sleeved on the liquid outlet valve seat, the lower end of the valve cover is movably abutted against the lower end of the liquid outlet valve seat, and the lower bottom surface of the valve cover is provided with a plurality of liquid outlet holes;
wherein, the lateral wall lowest department of collecting the room is equipped with the desorption liquid export that runs through, the lateral wall lowest department of desorption room is equipped with the desorption liquid import that runs through.
5. The method for detecting mycotoxin contamination in agricultural products as claimed in claim 4, wherein a sealing ring is arranged at a joint of the liquid outlet valve seat and the liquid running channel, and a sealing gasket is arranged at a joint of the desorption chamber and the collection chamber.
6. The method of detecting mycotoxin contamination in an agricultural product of claim 4, wherein the desorption fluid inlet is provided with a one-way valve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911391365.0A CN111122737B (en) | 2019-12-30 | 2019-12-30 | Detection method for mycotoxin pollution in agricultural products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911391365.0A CN111122737B (en) | 2019-12-30 | 2019-12-30 | Detection method for mycotoxin pollution in agricultural products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111122737A true CN111122737A (en) | 2020-05-08 |
| CN111122737B CN111122737B (en) | 2022-05-27 |
Family
ID=70504615
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911391365.0A Active CN111122737B (en) | 2019-12-30 | 2019-12-30 | Detection method for mycotoxin pollution in agricultural products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111122737B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102847512A (en) * | 2012-09-27 | 2013-01-02 | 江南大学 | Preparation method of rice husk based porous carbon fungaltoxin adsorbent |
| CN103760004A (en) * | 2013-12-31 | 2014-04-30 | 聚光科技(杭州)股份有限公司 | Solvent desorption device and method |
| CN106093254A (en) * | 2016-08-24 | 2016-11-09 | 上海市农业科学院 | A kind of enrichment purification method of zearalenones toxin |
| CN107144645A (en) * | 2017-04-11 | 2017-09-08 | 河南省粮油饲料产品质量监督检验中心 | It is a kind of while detecting the method for a variety of mycotoxins |
| CN107741462A (en) * | 2017-10-17 | 2018-02-27 | 江西省兽药饲料监察所 | A kind of detection method of 16 kinds of mycotoxins |
| CN110108822A (en) * | 2019-05-14 | 2019-08-09 | 苏州贞成分析仪器有限公司 | Aflatoxin sample extraction detection method and its purification pipette tips in a kind of edible vegetable oil |
-
2019
- 2019-12-30 CN CN201911391365.0A patent/CN111122737B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102847512A (en) * | 2012-09-27 | 2013-01-02 | 江南大学 | Preparation method of rice husk based porous carbon fungaltoxin adsorbent |
| CN103760004A (en) * | 2013-12-31 | 2014-04-30 | 聚光科技(杭州)股份有限公司 | Solvent desorption device and method |
| CN106093254A (en) * | 2016-08-24 | 2016-11-09 | 上海市农业科学院 | A kind of enrichment purification method of zearalenones toxin |
| CN107144645A (en) * | 2017-04-11 | 2017-09-08 | 河南省粮油饲料产品质量监督检验中心 | It is a kind of while detecting the method for a variety of mycotoxins |
| CN107741462A (en) * | 2017-10-17 | 2018-02-27 | 江西省兽药饲料监察所 | A kind of detection method of 16 kinds of mycotoxins |
| CN110108822A (en) * | 2019-05-14 | 2019-08-09 | 苏州贞成分析仪器有限公司 | Aflatoxin sample extraction detection method and its purification pipette tips in a kind of edible vegetable oil |
Non-Patent Citations (3)
| Title |
|---|
| 喻理等: "石墨烯吸附材料及其在真菌毒素检测中的应用", 《分析测试学报》 * |
| 曹冬梅等: "层状硅铝酸盐黏土对黄曲霉毒素的选择性化学吸附和脱毒机制", 《饲料广角》 * |
| 李珺沬等: "基于多巴胺－ 氧化石墨烯复合物的搅拌棒吸附萃取/高效液相色谱法测定食用油中黄曲霉毒素", 《分析测试学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111122737B (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111044643B (en) | Detection method of organic phosphate | |
| CN107543876A (en) | A kind of method that SPE liquid chromatography tandem mass spectrometry detects 9 kinds of estrogenic chemicalses in water body simultaneously | |
| CN110389185A (en) | The solid phase extraction method of a variety of steroid hormones and the detection method to the steroid hormone in a kind of saliva | |
| CN109342624A (en) | The method that Solid Phase Extraction pre-treatment combination LC-MS technology detects 15 kinds of antibiotic in aquaculture system simultaneously | |
| CN113702558A (en) | Method for detecting trace estrogen substances in water environment | |
| CN105181858B (en) | A kind of impurity absorption type decontaminating column and preparation method and application | |
| CN111122737B (en) | Detection method for mycotoxin pollution in agricultural products | |
| CN108845068B (en) | Detection method of pyrazine compounds in pan-fired green tea | |
| CN111983069B (en) | Detection method for determining phosphorus flame retardant and degradation product thereof in surface water | |
| CN111220722B (en) | Method for simultaneously determining 8 p-hydroxybenzoate compounds in soil | |
| CN108414643A (en) | The triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry | |
| CN108593832A (en) | LC-MS-MS methods that are a kind of while measuring six kinds of mycotoxins in corn | |
| CN109828072B (en) | Method for simultaneously detecting 16 biotoxins in brewing raw materials by using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer | |
| CN108828078B (en) | Method for determining inorganic anions in tobacco gene editing material | |
| CN108120791A (en) | A kind of method of three kinds of ochratoxin total amounts in Rapid Simultaneous Determination sample | |
| CN111679007A (en) | Method for simultaneously detecting 13 halogenated amide disinfection byproducts in drinking water | |
| CN102944635A (en) | Method for determining tris (2,3-dibromopropyl) phosphate content of water | |
| CN112986476A (en) | Method for measuring residual quantity of cuaminosulfate in vegetables and fruits | |
| CN112198242A (en) | Method for determining aflatoxins B1, B2, G1 and G2 in angelica sinensis by ultra-high performance liquid chromatography-mass spectrometry | |
| CN114354805A (en) | MOF-based SPME-DLLME-HPLC-MS method for detecting pesticide in food and application | |
| CN108519454B (en) | Pretreatment method for measuring various pesticide residues in tea and detection method thereof | |
| Meredith | [37] Isolation and characterization of fumonisins | |
| CN110208444B (en) | Method for detecting N-dimethyl nitrosamine in meat products | |
| CN219002020U (en) | Automatic reflux extraction instrument | |
| CN111239300B (en) | Method for extracting fatty acid in serum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |