CN112213420B - Method for rapidly determining multicomponent mycotoxins in beans and bean products - Google Patents
Method for rapidly determining multicomponent mycotoxins in beans and bean products Download PDFInfo
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- 231100000678 Mycotoxin Toxicity 0.000 title claims abstract description 67
- 239000002636 mycotoxin Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 34
- 244000046052 Phaseolus vulgaris Species 0.000 title claims abstract description 30
- 235000010627 Phaseolus vulgaris Nutrition 0.000 title abstract description 25
- 239000012629 purifying agent Substances 0.000 claims abstract description 28
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 5
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 14
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- 239000007788 liquid Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 235000013527 bean curd Nutrition 0.000 claims description 8
- 239000001509 sodium citrate Substances 0.000 claims description 7
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 7
- 239000003053 toxin Substances 0.000 claims description 7
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- 238000005119 centrifugation Methods 0.000 claims description 6
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- 244000068988 Glycine max Species 0.000 claims description 4
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 29
- 238000000605 extraction Methods 0.000 abstract description 23
- 238000004458 analytical method Methods 0.000 abstract description 16
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- IDGRYIRJIFKTAN-HTJQZXIKSA-N 15-acetyldeoxynivalenol Chemical compound C([C@@]12[C@]3(C)C[C@@H](O)[C@H]1O[C@@H]1C=C(C)C(=O)[C@@H](O)[C@@]13COC(=O)C)O2 IDGRYIRJIFKTAN-HTJQZXIKSA-N 0.000 abstract description 4
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- 235000021332 kidney beans Nutrition 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
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- 235000013339 cereals Nutrition 0.000 description 3
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- 230000005764 inhibitory process Effects 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
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- 238000004704 ultra performance liquid chromatography Methods 0.000 description 3
- 238000002137 ultrasound extraction Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 241000196324 Embryophyta Species 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
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- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
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Classifications
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for rapidly determining multicomponent mycotoxins in beans and bean products, which comprises the steps of preparing an extracting agent, a purifying agent A and a purifying agent B according to specific gravity, treating a sample by the extracting agent, the purifying agent A and the purifying agent B, and performing qualitative and quantitative analysis on the multicomponent mycotoxins by UPLC-MS/MS. The invention prepares the extracting agent and the purifying agent by adopting the chemical agent with lower laboratory cost, optimizes the proportion of the extracting agent and the purifying agent, can perform better purifying treatment on complex sample matrixes to remove sample analysis interference components, improves the accuracy and precision of measurement, has short operation time and high extraction and purifying effect, can finish quantitative analysis on mycotoxins of multiple components such as AFTB1, OTA, T-2, HT-2, ST, FB1, ZEN, NIV, DON, 3-AcDON, 15-AcDON and the like within 15min by combining the UPLC-MS/MS measuring technology of the invention, and has high measuring efficiency, quantitative sensitivity and small error.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for rapidly determining multicomponent mycotoxins in beans and bean products.
Background
Mycotoxins are toxic substances produced by metabolism of toxigenic fungi in a proper environment, and can pollute plant-derived agricultural products such as grains, beans, fruits, vegetables and the like, so that serious threat is generated to human health and livestock and poultry cultivation safety through the enrichment function of food chains, and mycotoxin pollution is regarded as the first of food-borne diseases by WHO and FAO.
In recent years, due to rapid development of mycotoxin sample analysis technology, in the actual analysis and detection process, the mycotoxin multicomponent analysis method has the problems of long time consumption, poor specificity, low recovery rate, complex operation, low detection efficiency, inaccurate quantification and qualitative determination and the like due to the complexity and diversity of mycotoxin sample analysis methods (a UPLC method, an HPLC-pre-column/post-column derivatization method, an ELISA method, a TLC method, a colloidal gold quantitative detection method, an immunoaffinity column-fluorescence photometry method and the like), sample pretreatment technologies (solvent extraction, liquid-liquid extraction, SPE technology and the like) and sample types (grains, beans, fruits, vegetables, marine products and the like).
Especially, bean crops are often polluted by various different mycotoxins in the growing and storing processes, and one fungus can generate various toxins, so that the mycotoxin pollution has the characteristics of complex components, wide pollution range, multiple types and large sample size. At present, no national standard method specially aiming at the analysis and determination of mycotoxin in beans and products thereof exists in China. The existing mycotoxin national standard analysis method is mainly used for analyzing grains, fruits, vegetables, feeds and the like, and beans and products thereof are often caused by the specificity of sample matrixes: soybeans, for example, often contain a large amount of protein and complexity: for example, in fermented bean curd, several tens of flavors are often added, and the above analysis method is used: UPLC method, HPLC-pre/post column derivatization method, ELISA method, TLC method, colloidal gold quantitative detection method, immunoaffinity column-fluorescence photometry method can not meet the requirement of bean and mycotoxin multicomponent analysis of products thereof, and the methods have respective defects on mycotoxin multicomponent analysis: such as UPLC and HPLC, require derivatization to analyze certain mycotoxins, increasing analysis costs and time due to detector limitations; the ELISA method, the colloidal gold quantitative detection method, the immunoaffinity column-fluorescence photometry and the like are limited by the specificity of antigen-antibody, so that only single or a plurality of mycotoxin components can be analyzed, the mycotoxin components can not be analyzed simultaneously, a special analysis kit is needed during the analysis, the cost is high, and the quantitative accuracy is poor.
Disclosure of Invention
Therefore, based on the background above, the invention provides a method for rapidly determining the multicomponent mycotoxins in beans and bean products, which can simultaneously analyze the multicomponent mycotoxins of beans and bean products in a short time, has short time consumption and low determination cost.
The invention adopts the technical scheme that:
a method for rapidly determining mycotoxin multiple components in legumes and bean products, comprising the steps of:
(1) Firstly, measuring an acetonitrile formate solution according to the volume ratio of 1-10%, and then measuring ultrapure water according to the volume ratio of 30-50% to prepare an acetonitrile formate-water solution extracting solution; na2SO4, naCl and sodium citrate are weighed according to the mass ratio of 4:4:1 and mixed to prepare a purifying agent A component; mgSO4, PSA and C18 are weighed according to the mass ratio of 3:2:1 and mixed to prepare a purifying agent B component;
(2) Weighing a certain amount of crushed sample to be measured, placing the crushed sample into a centrifuge tube, adding ultrapure water and an extracting agent, adding a purifying agent A, uniformly mixing for a period of time by using a mixer, extracting for a period of time by using an ultrasonic extractor, and freezing and centrifuging for a period of time at 8000-12000 rpm;
(3) Transferring the supernatant of the sample subjected to refrigerated centrifugation in the step (2) into another centrifuge tube, adding a purifying agent B component into the supernatant, uniformly mixing for a period of time by using a mixer, and refrigerated centrifuging for a period of time at 8000-12000 rpm;
(4) Filtering the supernatant of the sample obtained after the refrigerated centrifugation in the step (3) by using a 0.22 mu m organic filter membrane, and then analyzing for later use;
(5) And (3) qualitatively and quantitatively analyzing the mycotoxin multicomponent of the sample to be analyzed treated in the step (4) by adopting an ultra-high performance liquid chromatography-mass spectrometer (UPLC-MS/MS).
In a modification, the mycotoxins measured in step (5) include AFTB1, OTA, T-2, HT-2, ST, FB1 and ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON.
In a modification, toxins of AFTB1, OTA, T-2, HT-2, ST and FB1 were analyzed using ESI+ mode, and toxins of ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON were analyzed using ESI-mode.
In an improvement, the liquid chromatography column of UPLC-MS/MS in step (3) is a column comprising but not limited to C 18 Chromatographic column, said C 18 The chromatographic column has a length of 100-250 mm, wherein the positive ion adopts methanol-acetonitrile with a concentration of 0.01% -0.5% as a mobile phase, and the negative ion adopts ammonia water-methanol with a concentration of 0.01% -0.5% as a mobile phase.
In a modification, the mixer of the step (2) and the step (3) is a vortex mixer.
In a modification, the sample to be tested in the step (2) comprises, but is not limited to, soybeans, mung beans, kidney beans, small beans, kidney beans, peas, tofu and fermented bean curd.
The beneficial effects of the invention are as follows:
the method can be used for simultaneously measuring the mycotoxin multiple components in beans and bean products, and has the advantages of short time consumption, low cost and strong practicability. According to the invention, the extracting agent and the purifying agent are prepared by adopting the chemical agent with lower laboratory cost, the proportion of the extracting agent and the purifying agent is optimized, the prepared extracting agent and purifying agent can carry out better purifying treatment on complex sample matrixes so as to remove sample analysis interference components, the accuracy and the precision of measurement are improved, and the prepared extracting agent and purifying agent are low in cost, short in operation time and good in purifying effect; by combining the UPLC-MS/MS measuring technology, the multi-component quantitative analysis of mycotoxins such as AFTB1, OTA, T-2, HT-2, ST, FB1, ZEN, NIV, DON, 3-AcDON, 15-AcDON and the like can be completed within 15 minutes, and the measuring efficiency is high, the quantitative sensitivity is high, and the error is small.
Drawings
FIG. 1 is a total ion flow diagram of 6 mycotoxins experimentally examined in the example of the present invention, wherein FIG. 1 (1) is a total ion flow diagram of a negative ion compound; FIG. 1 (2) is a total ion flow diagram of a positive ion compound;
FIG. 2 is a chromatogram of 6 mycotoxins in an example of the present invention;
FIG. 3 is a graph of ion chromatograms of the addition of standard amounts of 6 mycotoxins in a hollow white sample according to an embodiment of the present invention;
FIG. 4 (1) is a standard curve of mycotoxin solvents for four negative ion compounds in examples of the present invention; FIG. 4 (2) is a standard curve of mycotoxin solvents for two cationic compounds in examples of the present invention;
FIG. 5 shows the extraction efficiency of 6 mycotoxins by different extraction reagents in an embodiment of the invention;
FIG. 6 shows the extraction efficiency of 6 mycotoxins according to the different extraction modes and extraction times of the present invention;
FIG. 7 shows the effect of the amounts of different components of scavenger A on recovery of 6 mycotoxins, wherein FIG. 7 (1) is Na in the example of the present invention 2 Influence of SO4 consumption on recovery rate; FIG. 7 (2) is the effect of NaCl usage on recovery in the examples of the present invention; FIG. 7 (3) is a graph showing the effect of sodium citrate usage on recovery in the examples of the present invention;
FIG. 8 shows the effect of different amounts of scavenger B on recovery of 6 mycotoxins, wherein FIG. 8 (1) shows the effect of MgSO4 amounts on recovery in the examples of the invention; FIG. 8 (2) is a graph showing the effect of PSA usage on recovery in an example of the invention; FIG. 8 (3) is a diagram of embodiment C of the present invention 18 Influence of the amount used on recovery.
Detailed Description
The invention is described in further detail below by way of specific embodiments:
example 1: a method for rapidly determining multicomponent mycotoxins in beans and bean products comprises the following steps:
(1) Firstly, measuring an acetonitrile formate solution according to the volume ratio of 1-10%, and then measuring ultrapure water according to the volume ratio of 30-50% to prepare an acetonitrile formate-water solution extracting solution; na is weighed according to the weight ratio of 4:4:1 2 SO 4 Mixing NaCl and sodium citrate to prepare a component A of the purifying agent; mgSO was weighed at a weight ratio of 3:2:1 4 、PSA、C 18 And mixing the components to prepare a purifying agent B component;
(2) Weighing 5.0g (0.01 g accurately) of crushed sample to be tested, putting the crushed sample into a 50mL centrifuge tube, adding ultrapure water and an extracting agent, adding the purifying agent A component, uniformly mixing for 2-3min by a mixer, extracting for 5-15min by ultrasonic wave, and freezing and centrifuging for a period of time at 8000-12000 r/min;
(3) Transferring the supernatant of the sample of the centrifugal tube subjected to the refrigerated centrifugation in the step (2) into another centrifugal tube, adding a purifying agent B component into the supernatant, uniformly mixing for 2-5min by a mixer, and refrigerated centrifuging for 5-10min at 8000-12000 rpm;
(4) Filtering the supernatant of the sample subjected to refrigerated centrifugation in the step (3) by adopting a 0.22 mu m organic filter membrane, and then analyzing for later use;
(5) And (3) carrying out qualitative and quantitative analysis on the multi-component mycotoxin of the sample to be analyzed after the treatment in the step (4) by adopting UPLC-MS/MS.
Mycotoxins determined in step (5) include AFTB1, OTA, T-2, HT-2, ST, FB1 and ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON.
The toxins of AFTB1, OTA, T-2, HT-2, ST and FB1 employ ESI + Pattern analysis, toxins of ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON using ESI - And (5) pattern analysis.
The liquid chromatographic column of UPLC-MS/MS in the step (3) is a C18 chromatographic column, including but not limited to, the length of the C18 chromatographic column is 100mm-250mm, wherein positive ions adopt methanol-acetonitrile with the concentration of 0.01% -0.5% as a mobile phase, and negative ions adopt ammonia water-methanol with the concentration of 0.01% -0.5% as a mobile phase.
The mixers of the step (2) and the step (3) are vortex mixers.
The sample to be tested in step (2) includes, but is not limited to, soybeans, mung beans, kidney beans, small beans, kidney beans, peas, tofu, fermented bean curd and other tofu products.
The experimental auxiliary equipment used in the embodiment mainly comprises a ten-thousandth balance (Metrehler, model: B714929749), a ten-thousandth balance (Metrehler, model: XSE 105), an ultrasonic cleaner (Zhongchen technology, model: SD 290H), a high-speed refrigerated centrifuge (Kate laboratory instrument, model: TCL 16M), and a constant-temperature vibration extractor (Lebertiaceae, model: LW 20); the main detection instrument used in the experiment is a liquid chromatography-mass spectrometer (AB SCIEX, model: TRIPLE QUAD 4500), and the control parameters of the liquid chromatography-mass spectrometer are as follows:
1. liquid chromatography parameters
1) Liquid chromatographic column: moon, UHPLC AQ-C18 column (column length 100mm, column inner diameter 2.1mm, filler particle size 1.8 μm), or equivalent column.
2) Chromatographic column temperature: 30 ℃.
3) Sample injection amount: 2 mu L of positive ion compound (ESI+) and 3 mu L of negative ion compound (ESI-) are adopted, and if the positive and negative switching scanning mode is adopted, the sample injection amount can be uniformly selected to be 5 mu L.
4) Flow rate: 0.3mL/min.
5) Mobile phase: phase A: 0.05% formic acid aqueous solution (esi+)/0.05% ammonia (ESI-); and B phase: acetonitrile (esi+)/methanol (ESI-). If mycotoxin detection is carried out simultaneously in the positive and negative ion switching mode, phase A: 0.1% formic acid+2 mmol/L ammonium acetate solution; and B phase: acetonitrile.
6) The mycotoxin standard solution (with the concentration of 100 ng/mL) is used for chromatographic separation through a C18 chromatographic column, the peak outlet time, the peak broadening and the ion response value of a chromatographic peak are observed, the optimized elution program is adjusted, and the optimized gradient elution program is shown in tables 1 and 2.
Table 1: positive ion mode liquid chromatography gradient elution procedure
Table 2: anion mode liquid chromatography gradient elution procedure
2. Mass spectral parameters
1) Ion source: an electrospray ion source.
2) Mass spectrometry scan mode: multiple reaction monitoring mode (MRM).
3) Ionization voltage: positive ion mode (eis+) +5500V/negative ion mode (EIS-) -4500V.
4) Curtain gas (CUR): 35psi.
5) Ion source temperature: 500 ℃.
6) Spray gas (GS 1): 50psi.
7) Auxiliary heating gas (GS 2): 50psi.
8) Sampling by adopting a needle pump constant-current mode, respectively carrying out primary mass spectrum full scanning (Q1 Scan) on standard solutions (the concentration is 100 ng/mL) of 11 mycotoxins, and determining the mass-to-charge ratios of various mycotoxin parent ions; the fragment ions generated by nitrogen collision are subjected to secondary mass spectrometry (Product Ion Scan), the fragment ions with the largest response value are selected as quantitative ions, the secondary response ions are used as sub-ions, and the declustering voltage (DP) and the Collision Energy (CE) are optimized, so that the optimal mass spectrometry parameters are shown in tables 3 and 4.
Table 3: mass spectrum parameters of 5 negative ion mode mycotoxins
Note that: * Indicating quantitative daughter ions.
Table 4: mass spectrum parameters of 6 positive ion mode mycotoxins
Note that: * Indicating quantitative daughter ions.
3. Preparing mycotoxin mixing standard with concentration of 100ng/mL, and performing MRM mass spectrum scanning by adopting an optimized liquid chromatography mass spectrum method to obtain total ion flow diagrams and chromatograms of 6 mycotoxins, wherein the total ion flow diagrams and chromatograms are shown in figures 1 and 2 respectively.
4. The blank sample is used as a matrix, 6 mycotoxins are added according to the low concentration level, the medium concentration level and the high concentration level, and the addition concentrations (a), (b) and (c) are 5, 50 and 200 mug/kg respectively, as shown in figure 3.
5. Preparing a mixed standard curve of 6 mycotoxins respectively by using a blank solvent (20% methanol aqueous solution), wherein the concentration points of the standard curve of the negative ion compound are 10, 20, 30, 50, 100, 150 and 200ng/mL; the standard curve concentration points of the positive ion compound are 1, 2, 5, 10, 20, 50, 100 and 200ng/mL, and the obtained standard curves are shown in fig. 4 (1) and fig. 4 (2).
6. Sample extraction method development
1) Investigation of extraction solvent and extraction mode
(1) Investigation of extraction solvent: and selecting blank samples, respectively examining the extraction efficiency of different extracting solutions on 6 mycotoxins through the mycotoxin adding recovery rate (the standard concentration is 100 mug/kg), taking 3 parallel samples for each standard sample, taking an average value to calculate the recovery rate, wherein the recovery rate is 80% -120%, the extraction efficiency is better, and the result shows that the overall extraction effect of 1% of acetonitrile formate on the mycotoxins is better, as shown in figure 5.
(2) Investigation of extraction modes: a blank sample is selected, extraction time (10, 20, 30 and 60 min) and extraction mode (vibration extraction and ultrasonic extraction) are respectively examined through mycotoxin adding recovery rate (100 mug/kg), extraction efficiency of 6 mycotoxins is respectively examined, each labeled sample is prepared into 3 parallel samples, recovery rate is calculated, the recovery rate is 80% -120%, the result shows that the vibration extraction for 10min and the ultrasonic extraction for 10min can be better, and the ultrasonic extraction for 10min is adopted for simplicity and convenience, as shown in figure 6.
2) Inspection of the amount of the purifying agent A
The experimental scheme respectively examines Na through the mycotoxin addition recovery rate (the standard concentration is 100 mug/kg) 2 The higher the recovery rate of SO4, naCl and sodium citrate, the better the extraction effect, and Na is selected according to the recovery rate 2 The dosage of SO4, naCl and sodium citrate is comprehensively considered Na 2 The amounts of SO4, naCl and sodium citrate are respectively 4g, 4g and 1g, and the examination results are shown in the accompanying figures 7 (1), 7 (2) and 7 (3).
3) Investigation of the amount of the purifying agent B
According to the experimental scheme, the recovery rate of the mycotoxin addition (100 mug/kg) is adopted to respectively inspect the MgSO4, the PSA and the C18, the recovery rate is 80% -120%, the extraction effect is good, the consumption of the MgSO4, the PSA and the C18 is selected according to the recovery rate, the consumption of 600mg, 400mg and 200mg are comprehensively considered, and the inspection results are shown in the accompanying figures 8 (1), 8 (2) and 8 (3).
7. The matrix effect, the linear relation, the detection limit, the accuracy and the precision of the method are verified and inspected, and the result is as follows:
1) Investigation of matrix effects
The purifying agent A, the purifying agent B and the extracting agent developed by the scheme are adopted to extract blank matrix solution, a standard curve is prepared, the matrix effect is evaluated by the ratio K of the slope of the matrix matching standard curve and the slope of the organic solvent standard curve, if the K value is between 0.9 and 1.1, the matrix effect is not obvious, and if the K value is more than 1.1, the matrix effect is the matrix enhancement effect, and if the K value is less than 0.9, the matrix inhibition effect is the matrix inhibition effect, 6 mycotoxins in 8 matrixes are respectively examined by experiments, and other mycotoxin matrix effects are not obvious except DON which is expressed as the matrix inhibition effect, and in order to ensure the accuracy of detection results, the matrix effect can be eliminated by an internal standard method when the DON is measured, and the examination results are shown in Table 5.
TABLE 5-1:15 matrix effect investigation of AcDON in 8 matrices
TABLE 5-2:3-matrix Effect investigation of AcDON in 8 matrices
TABLE 5-3 investigation of the matrix effect of DON in 8 matrices
TABLE 5-4 investigation of matrix effects of ZEN in 8 matrices
Tables 5-5: investigation of matrix Effect of OTA in 8 matrices
Tables 5-6 matrix effect investigation of AFTB1 in 8 matrices
2) Linear relation, detection limit and quantitative limit investigation
As shown in Table 6, 15-AcDON and 3-AcDON, DON, ZEN all have good linear relationship in the range of 10-300 mug/L, OTA and AFTB1 have good linear relationship in the range of 1-200 mug/L, and the correlation coefficient R is not less than 0.9992. The method is used for measuring a blank sample with low concentration and standard deviation of detection results for 10 times, the standard deviation of the detection results is s, the blank value of the sample is +4.65s as a detection Limit (LOD), 3 times of LOD is used as a lower limit of quantification (LOQ), the detection limit of 6 mycotoxins in different matrixes is 0.3-16.3 mug/kg, and the detection limit meets the requirements of GB13078-2017 limit.
Table 6: linear range, linear equation, correlation coefficient (r), detection limit and quantification limit of 6 mycotoxins
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3) Accuracy and precision investigation
According to the invention, 6 mycotoxins are respectively examined for accuracy by adopting different adding standard amounts (low, medium and high) according to different sample matrixes, wherein the adding levels (low, medium and high) of 15-AcDON, 3-AcDON, DON and ZEN 4 mycotoxins are respectively 20, 100 and 400 mug/kg, the adding levels (low, medium and high) of OTA and AFTB1 are respectively 2, 10 and 100 mug/kg, each adding level is measured for 3 times, the average value is taken, the average recovery rate is 81.5% -119%, and the results are shown in Table 7. The method also comprises the steps of selecting different matrixes to carry out a labeling experiment, repeatedly measuring each sample for 7 times, and calculating the relative standard deviation to obtain the precision of 0.53-2.51%, wherein the result is shown in Table 8.
Table 7: accuracy investigation of 6 mycotoxins in different matrices
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Table 8: precision investigation of 6 mycotoxins in different matrices
The extractant, the purifying agent A and the purifying agent B prepared by the invention have simple preparation process, strong universality, low price, short extraction and purification time consumption and environmental friendliness, and can simultaneously satisfy the extraction and purification of multiple types of samples. The UPLC-MS/MS method has strong selectivity, good sample separation degree and accurate quantitative and qualitative properties, and can meet the confirmation requirement of the analysis method by verifying and inspecting the matrix effect, the linear relation, the detection limit, the accuracy and the precision of the method.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (2)
1. A method for rapidly determining multicomponent mycotoxins in legumes and legume products, comprising the steps of:
(1) Firstly, measuring an acetonitrile formate solution according to the volume ratio of 1-10%, and then measuring ultrapure water according to the volume ratio of 30-50% to prepare an acetonitrile formate-water solution extracting solution; na is weighed according to the mass ratio of 4:4:1 2 SO 4 Mixing NaCl and sodium citrate to prepare a purifying agent A component; mgSO is weighed according to the mass ratio of 3:2:1 4 、PSA、C 18 And mixing the components to prepare a purifying agent B component;
(2) Weighing a certain amount of crushed sample to be measured, placing the crushed sample into a centrifuge tube, adding ultrapure water and an extracting agent, adding a purifying agent A, uniformly mixing for a period of time by using a mixer, extracting for a period of time by using an ultrasonic extractor, and freezing and centrifuging for a period of time at 8000-12000 rpm;
(3) Transferring the supernatant of the sample subjected to refrigerated centrifugation in the step (2) into another centrifuge tube, adding a purifying agent B component into the supernatant, uniformly mixing for a period of time by using a mixer, and refrigerated centrifuging for a period of time at 8000-12000 rpm;
(4) Filtering the supernatant of the sample obtained after the refrigerated centrifugation in the step (3) by using a 0.22 mu m organic filter membrane, and then analyzing for later use;
(5) Carrying out qualitative and quantitative analysis on the mycotoxin multicomponent of the sample to be analyzed treated in the step (4) by adopting an ultra-high performance liquid chromatography-mass spectrometer; the liquid chromatographic column of the ultra-high performance liquid chromatography mass spectrometer is C 18 Chromatographic columnThe C is 18 The length of the chromatographic column is 100mm-250mm, wherein formic acid with the concentration of 0.01% -0.5% is adopted as a mobile phase A and acetonitrile is adopted as a mobile phase B in a positive ion mode, and the liquid chromatographic gradient elution program in the positive ion mode is shown in table 1; the anion mode adopts ammonia water with the concentration of 0.01% -0.5% as a mobile phase A and methanol as a mobile phase B, and the liquid chromatographic gradient elution program of the anion mode is shown in Table 2;
TABLE 1 Positive ion mode liquid chromatography gradient elution procedure
TABLE 2 anion mode liquid chromatography gradient elution procedure
Mycotoxins determined in step (5) include AFTB1, OTA, T-2, HT-2, ST, FB1 and ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON; toxins of AFTB1, OTA, T-2, HT-2, ST and FB1 were analyzed using esi+ mode, and toxins of ZEN, NIV, DON, 3-Ac-DON and 15-Ac-DON were analyzed using ESI-mode.
2. The method according to claim 1, wherein the sample to be tested in step (2) includes, but is not limited to, soybean, mung bean, kidney bean, pea, bean curd, fermented bean curd.
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| CN106770836A (en) * | 2017-01-16 | 2017-05-31 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of at the same detect cereal in various mycotoxins method |
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