CN115452990A - Method for detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven - Google Patents
Method for detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven Download PDFInfo
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- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 title claims abstract description 54
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 46
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 44
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 title claims abstract description 33
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 title claims abstract description 33
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 title claims abstract description 33
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
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- G01N30/08—Preparation using an enricher
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Abstract
The invention relates to a method for detecting mycotoxin in food and medicine, in particular to a method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven by immunoaffinity column purification, post-column photochemical derivatization and high performance liquid chromatography. According to the method, XBridge Phenyl columns are used, acetonitrile is used as a mobile phase A,0.2% acetic acid solution is used as a mobile phase B, gradient elution is carried out, the flow rate is 1mL/min, and the column temperature is 35 ℃; the variable fluorescence detection wavelength is 0-20 min, the excitation wavelength is 365nm, and the emission wavelength is 450nm (detecting aflatoxins G2, G1, B2 and B1); 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm (detecting zearalenone); 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm (detecting ochratoxin A). The method is simple, convenient and rapid, has good purification effect, has various technical indexes meeting the requirements of mycotoxin detection, and can meet the requirement of limited detection for simultaneously determining aflatoxin, zearalenone and ochratoxin A in the medicated leaven by 1-time sample injection.
Description
Technical Field
The invention relates to a method for detecting mycotoxin in food and medicine, in particular to a method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven by immunoaffinity column purification, post-column photochemical derivatization and high performance liquid chromatography.
Background
Aflatoxins (AFT) are difuranocyclic toxoids produced by some strains of aspergillus flavus and aspergillus parasiticus. There are about 20 derivatives, which are named as B1, B2, G1, G2, M1, M2, GM, P1, Q1, toxol, etc. Among them, B1 has the highest toxicity and the highest carcinogenicity. After animals eat the aflatoxin-contaminated feed, trace amounts of toxins can be detected in the liver, kidneys, muscles, blood, milk and eggs. Aflatoxins and producing strains thereof are widely distributed in nature, some strains produce more than one type of aflatoxin, and strains which do not produce any type of aflatoxin also exist in aspergillus flavus. Aflatoxins mainly contaminate grains and oils and their products, as well as various vegetable and animal foods. The aflatoxin-producing aspergillus flavus is easy to parasitize and produce toxins in cereal crops, oil crop seeds and processing byproducts thereof with high water content (the water content is lower than 12 percent and cannot be propagated), so that the aflatoxin-producing aspergillus flavus can mildew and deteriorate, and people eat the food or the processing byproducts thereof by mistake and absorb the toxins into human bodies through alimentary canals to be poisoned.
Zearalenone (Zearalenone), also known as the F-2 toxin, was first isolated from maize with head blight. Zearalenone mainly contaminates corn, wheat, rice, barley, millet, oats and other grains. The zearalenone has strong heat resistance, and can be completely destroyed after being treated at 110 ℃ for 1 hour. Zearalenone has estrogen-like effect, can cause acute and chronic poisoning of animals, cause abnormal and even death of animal reproductive function, and can cause huge economic loss to animal farms. Zearalenone is a metabolite of zearalenone.
Aspergillus toxin (aspertoxin), a compound produced by the metabolism of Aspergillus flavus. Metabolites or secondary metabolites produced by koji molds, which are generally found in grains that have become moldy, may cause serious damage such as teratogenicity, mutagenicity, and carcinogenesis once they enter the human body through the food chain, and thus are receiving much attention in global food safety. Toxicology studies indicate that ochratoxin A has strong hepatotoxicity and nephrotoxicity, and has teratogenic, mutagenic and carcinogenic effects. Ochratoxin A is widely contained in grains and grain products, spices and coffee beans, and seriously harms human health.
The medicated leaven is prepared by adding flour or bran into herba Polygoni Hydropiperis, herba Artemisiae Annuae, and semen Armeniacae amarum, mixing, and fermenting. During the processing, flour or bran is added, and mycotoxins such as aflatoxin, zearalenone, ochratoxin A and the like can be brought in. In addition, mycotoxins such as aflatoxin have been detected from various traditional Chinese medicinal materials at present. With the increasing pollution of mycotoxins to traditional Chinese medicinal materials, a method for efficiently and accurately detecting trace aflatoxins, zearalenone, ochratoxin A and other mycotoxins in the medicated leaven needs to be established, and technical support is provided for the quality of the medicated leaven. However, the prior detection methods for mycotoxins such as aflatoxin, zearalenone, ochratoxin A and the like in food and medicine mainly comprise a thin-layer chromatography method, an enzyme-linked immunosorbent assay method, an immunoaffinity column purification fluorescence photometry method, an immunoaffinity column purification high-performance liquid chromatography method, a multifunctional column chromatography purification high-performance liquid chromatography method, a mass spectrometry method and the like. The former 3 methods have poor quantitative accuracy, while the latter 2 methods can only detect a single kind of mycotoxin, and the mass spectrometry has high requirements on cost and personnel of enterprises.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in the medicated leaven, the method adopts immunoaffinity column purification, photochemical derivatization after column and high performance liquid chromatography to simultaneously detect various mycotoxins, the method is simple, convenient and rapid, the purification effect is good, each technical index meets the requirement of mycotoxin detection, and the method can meet the limited detection requirement of simultaneously detecting aflatoxin, zearalenone and ochratoxin A in the medicated leaven by 1-time sample injection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for simultaneously detecting aflatoxins, zearalenone and ochratoxin A in medicated leaven is disclosed, wherein the aflatoxins are aflatoxins B1, B2, G1 and G2; the method comprises the following steps:
1) Sample extraction
Precisely weighing 5g of a test sample, precisely adding 50ml of 80% methanol with volume percentage concentration, performing ultrasonic treatment for 40min, transferring to a 50ml centrifuge tube, and performing 6000-turn centrifugation for 10min; precisely measuring 10ml of supernatant, putting the supernatant into a 50ml measuring flask, diluting the supernatant to a scale with water, shaking up, centrifuging for 10min at 6000 rpm, measuring 25ml of supernatant, eluting the supernatant through an immunoaffinity column, adding 20ml of water after the elution is finished, eluting again, discarding the eluent, extruding the water in the column, adding 2ml of methanol for elution, collecting the eluent, and shaking up;
2) High performance liquid chromatography assay
The device adopts Shimadzu high performance liquid chromatograph and photochemical derivatization instrument manufactured by Germany LC-Tech company
Phenyl column using acetonitrile as mobile phase A and 0.1% volume percentage concentration acetic acid solution as mobile phase B, gradient eluting with flow rate of 1mL/min and column temperature of 35 deg.C; the gradient elution was as follows:
detecting the aflatoxins G2, G1, B2, B1: excitation wavelength is 365nm and emission wavelength is 450nm for 0-20 min; detecting zearalenone: 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm;
detection of ochratoxin a: 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm.
Preferably, the method further comprises the steps of:
precisely measuring a proper amount of reference substance, adding methanol for dilution to obtain the reference substance, wherein the sample volume of the reference substance is 2, 5, 10, 15 and 20 mu l, injecting the reference substance into a liquid chromatograph, recording peak areas, taking the concentration (X, mu g and mL) of each reference substance as a horizontal coordinate, taking a peak area Y as a vertical coordinate, and obtaining the regression equation of each component as follows:
| composition (I) | Regression equation | r^2 |
| G2 | y＝2361.2x+31.366 | 0.9999 |
| G1 | y＝777.22x-30.634 | 0.9999 |
| B2 | y＝3239.5x+106.73 | 0.9998 |
| B1 | y＝959.6x+31.829 | 0.9991 |
| Zearalenone | y＝14109x+77.512 | 0.9992 |
| Ochratoxin A | y＝223.58x-70.38 | 0.9995 |
Preferably, the preparation method of the aflatoxin reference substance comprises the following steps: control solution lot No. 610001-202107, michem Acad, B 1 1.08μg/mL、B 2 Concentration 0.33. Mu.g/mL, G 1 The concentration is 1.01 mu G/mL, G 2 Precisely measuring 1mL of reference substance solution at a concentration of 0.33 mug/mL, placing into a 20mL measuring flask, diluting to scale with methanol to obtain stock solution; precisely measuring 0.4ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
Preferably, the preparation method of the zearalenone control product is as follows: reference substance solution lot No. 610022-202101, zhongzhong hospital, 10.1. Mu.g/ml, precisely measuring 1ml of reference substance solution, placing into a 10ml measuring flask, diluting to scale with methanol, and using as stock solution; precisely measuring 1ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
Preferably, the ochratoxin A reference substance is prepared by the following method: the batch number of the reference substance solution 2B00G12, purpura, 100.9 mu G/ml, precisely measuring 0.1ml of the reference substance solution, placing the reference substance solution in a 100ml measuring flask, and diluting the reference substance solution to a scale with methanol to obtain a stock solution; precisely measuring 2.5ml of the stock solution, placing into a 100ml measuring flask, diluting with methanol to scale, and shaking.
Preferably, the method further comprises the steps of:
taking one part of the mixed standard solution of the reference substance, injecting samples for 0h, 4h, 8h, 12h, 16h, 20h and 24h respectively, wherein the sample injection amount is 20 mu l, measuring the peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A respectively, and calculating RSD, wherein the results are as follows:
preferably, the method further comprises the steps of:
taking 5.0G of medicated leaven powder, precisely weighing 3 parts of medicated leaven powder, respectively placing the medicated leaven powder into 500mL conical flasks with stoppers, respectively adding 0.8mL of aflatoxin reference stock solution, 1mL of zearalenone reference stock solution and 0.2mL of ochratoxin reference stock solution into the conical flasks with stoppers, operating according to the method described in the application, introducing 10 muL of sample for measurement, and calculating the recovery rate, wherein the average sample addition recovery rates (n = 6) of G2, G1, B2 and B1, zearalenone and ochratoxin A are respectively as follows: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD of 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
The method adopts the technical scheme that the immunoaffinity column purification, the post-column photochemical derivatization and the high performance liquid chromatography are adopted for simultaneous detection, the method is simple, convenient and quick, the purification effect is good, all technical indexes meet the requirements of mycotoxin detection, and the requirement of limited detection that aflatoxin, zearalenone and ochratoxin A in the medicated leaven can be simultaneously detected by 1 sample injection can be met.
Drawings
FIG. 1 is a liquid chromatogram of a mixed reference substance of aflatoxin, zearalenone and ochratoxin A.
FIG. 2 is a liquid chromatography of a test sample L05.
Detailed Description
1. Test method
1.1 instruments
Shimadzu high performance liquid chromatograph, photochemical derivatization instrument is Germany LC-Tech company; a high speed refrigerated centrifuge, a constant temperature ultrasonic cleaner, METTLER TOLEDO XP26 (parts per million analytical balance), XP204 (one hundred thousand analytical balance); immunoaffinity column-multiple toxins (aflatoxins, zearalenone, ochratoxin a) were produced by Qingdao Pop bioengineering, inc.
1.2 reagents
Acetonitrile, methanol (chromatographically pure); acetic acid (guaranteed reagent); aflatoxin (B1, B2, G1, G2) mixed reference, zearalenone, and ochratoxin A reference; the experimental water was ultrapure water prepared from Millipore.
1.3 reagent
Medicated leaven is provided by Zhejiang tung juntang Chinese herbal pieces ltd.
1.4 chromatographic conditions and System Adaptation
A chromatographic column:
phenyl column (4.6X 250mm,5 μm) was eluted with a gradient of acetonitrile-0.1% acetic acid solution (30). The flow rate is 1mL/min, and the column temperature is 35 ℃; the variable fluorescence detection wavelength is 0-20 min, the excitation wavelength is 365nm, and the emission wavelength is 450nm (detecting aflatoxins G2, G1, B2 and B1); 20.01-25 min, the excitation wavelength is 274nm, and the emission wavelength is 460nm (detecting zearalenone); 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm (detecting ochratoxin A).
Sample amount of the reference: 2.5, 10, 15 and 20 mul, sample amount of a sample: 20 μ l.
1.5 solution preparation
1.5.1 control solutions
(1) Aflatoxin control (batch No. 610001-202107, zhongzhong college, B) 1 1.08μg/mL、B 2 Concentration 0.33. Mu.g/mL, G 1 Concentration 1.01. Mu.g/mL, G 2 Concentration 0.33. Mu.g/mL): 1ml of the control solution was precisely measured and placed in a 20ml measuring flask, and diluted to the scale with methanol to serve as a stock solution. Precisely measuring 0.4ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
(2) Zearalenone control (run No. 610022-202101, midhouse, 10.1 μ g/ml): 1ml of the control solution was precisely measured and placed in a 10ml measuring flask, and diluted to the scale with methanol as a stock solution. Precisely measuring 1ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
(3) Ochratoxin a control (batch No. 2B00G12, promapontan, 100.9 μ G/ml), 0.1ml of the control solution was precisely measured and placed in a 100ml measuring flask, diluted to the scale with methanol, and used as stock solution. Precisely measuring 2.5ml of the stock solution, placing into a 100ml measuring flask, diluting with methanol to scale, and shaking.
1.5.2 test article solution
Precisely weighing 5g of test sample, precisely adding 50ml of 80% methanol, performing ultrasonic treatment for 40min, transferring into a 50ml centrifuge tube, and centrifuging for 10min (6000 rpm). Precisely measuring 10ml of supernatant, putting the supernatant into a 50ml measuring flask, diluting the supernatant to a scale with water, shaking up, centrifuging for 10min (6000 revolutions), measuring 25ml of supernatant, eluting the supernatant through an immunoaffinity column, adding 20ml of water after the elution is finished, eluting again, discarding the eluent, squeezing out the water in the column, adding 2ml of methanol for elution, collecting the eluent, and shaking up.
Methodology review
2.1 Standard Curve
Precisely measuring appropriate amount of reference substance, diluting with methanol to obtain reference substance with sample amount of 2, 5, 10, 15, 20 μ l, injecting into liquid chromatograph, recording peak area, taking concentration (X, μ g. ML) of each reference substance as abscissa, taking peak area Y as ordinate, and making regression equation of each component as shown in Table 1.
Table 1 regression equation, correlation coefficient and r (n = 6)
| Composition (A) | Regression equation | r^2 |
| G2 | y＝2361.2x+31.366 | 0.9999 |
| G1 | y＝777.22x-30.634 | 0.9999 |
| B2 | y＝3239.5x+106.73 | 0.9998 |
| B1 | y＝959.6x+31.829 | 0.9991 |
| Zearalenone | y＝14109x+77.512 | 0.9992 |
| Ochratoxin A | y＝223.58x-70.38 | 0.9995 |
2.2 control stability test
And taking one part of the mixed standard solution of the reference substance, injecting samples for 0h, 4h, 8h, 12h, 16h, 20h and 24h respectively, wherein the injection amount is 20 mu l, measuring the peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A respectively, and calculating RSD, wherein the results are shown in a table 2.
TABLE 2 control stability test
| Composition (A) | RSD％ |
| G2 | 1.48 |
| G1 | 0.85 |
| B2 | 1.82 |
| B1 | 2.38 |
| Zearalenone | 3.19 |
| Ochratoxin A | 1.24 |
2.3 sample application recovery test
Taking 5.0G of medicated leaven powder, 3 parts in total, precisely weighing, respectively placing into 500mL conical flasks with stoppers, respectively adding 0.8mL of aflatoxin reference stock solution, 1mL of zearalenone reference stock solution and 0.2mL of ochratoxin reference (precisely measuring 0.1mL of reference stock solution, adding 80% methanol to dilute to 50mL of scale mark) according to the method of 1.5.2, carrying out sample injection of 10 mu L under the chromatographic condition of 1.4, and calculating the recovery rate, wherein the average sample injection recovery rates (n = 6) of G2, G1, B2 and B1, zearalenone and ochratoxin A are respectively: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD of 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
Results of three
3.1 assay of samples
In the application, 16 medicated leaven samples produced by 10 companies are respectively taken, test solution is prepared according to the method of 1.5.2, content measurement is carried out according to the chromatographic condition of 1.4, and the content of each component in the medicated leaven is calculated by adopting an external standard method, and the result is shown in table 3.
TABLE 3 measurement results of Massa Medicata Fermentata (. Mu.g/kg)
Discussion of the fourth
4.1. Extraction method
On a chromatographic column:
phenyl column (4.6X 250mm,5 μm) was eluted with gradient using acetonitrile-0.1% acetic acid solution (30). The flow rate is 1mL/min, and the column temperature is 35 ℃; the variable fluorescence detection wavelength is 0-20 min, the excitation wavelength is 365nm, and the emission wavelength is 450nm (detecting aflatoxins G2, G1, B2 and B1); 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm (detecting zearalenone); 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm (detecting ochratoxin A).
TABLE 4 gradient elution procedure
(1) When the extraction solution was changed to 70% acetonitrile, the recovery rates were calculated, and the results of sample addition recovery rates of G2, G1, B2, B1, zearalenone and ochratoxin a were: 70.96%, 110.56%, 106.41%, 104.32%, 80.84%, 0.0%.
(2) When the extraction solution was changed to 80% acetonitrile, the recovery rates were calculated, and the results of sample addition recovery rates of G2, G1, B2, B1, zearalenone and ochratoxin a were: 96.80%, 114.00%, 111.14%, 103.61%, 97.73%, 0.0%.
4.2. Immunoaffinity column investigation
Keeping the same with the chromatographic conditions of item 4.1, changing immunoaffinity column-multiple toxins (aflatoxin, zearalenone, ochratoxin A) into immunoaffinity column-ochratoxin A.
(1) When the extraction solution was 80% methanol, the recovery rate was calculated, and the sample recovery rate of ochratoxin a was 41.99%.
(2) When the extraction solution was 80% acetonitrile, the recovery rate was calculated, and the sample recovery rate of ochratoxin a was 46.81%.
4.3. Chromatographic column investigation
ZORBAX C18 (4.6X 250mm,5 μm), EXTEND C18 (4.6X 250mm,5 μm), phenomenex C18 (4.6X 250mm,5 μm) were compared at the previous stage,
phenyl column (4.6X 250mm,5 μm) and other chromatographic columns, it was found that the first three columns exhibited erratic retention time of 4 components of aflatoxin with increasing number of needle insertions during the experiment, and finally, some overlap was observed, so that the degree of separation was not satisfactory. While
Phenyl column (4.6X 250mm,5 μm) remained time stable throughout the experimentThe peak pattern was good.
4.4. Selection of mobile phase
Comparing organic phase methanol and acetonitrile, inorganic phase water, 0.1% phosphoric acid, 0.1% acetic acid, 0.2% acetic acid and 0.5% acetic acid, it is found that methanol and 0.1% acetic acid solution can not separate target peak effectively, and acetonitrile and acid solution can separate target component. The research is carried out on acid solutions of different types and different proportions and a reference substance of the same concentration, different acid and acid concentrations are found to have certain influence on peak areas, and finally, the gradient elution of 0.1 percent acetic acid and acetonitrile is determined to be selected as a mobile phase, and the comparison result is shown in the following table 5.
TABLE 5 peak areas of different mobile phases
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven comprises the following steps of (1) detecting aflatoxins B1, B2, G1 and G2; the method is characterized by comprising the following steps:
1) Sample extraction
Precisely weighing 5g of a test sample, precisely adding 50ml of 80% methanol with volume percentage concentration, performing ultrasonic treatment for 40min, transferring the sample into a 50ml centrifuge tube, and performing 6000-turn centrifugation for 10min; precisely measuring 10ml of supernatant, putting the supernatant into a 50ml measuring flask, diluting the supernatant to a scale with water, shaking up, centrifuging for 10min at 6000 rpm, measuring 25ml of supernatant, eluting the supernatant through an immunoaffinity column, adding 20ml of water after the elution is finished, eluting again, discarding the eluent, extruding the water in the column, adding 2ml of methanol for elution, collecting the eluent, and shaking up;
2) High performance liquid chromatography assay
The measuring equipment adopts Shimadzu high performance liquid chromatograph and photochemical derivatization instrument manufactured by Germany LC-Tech company
The Phenyl column uses acetonitrile as a mobile phase A and acetic acid solution with the concentration of 0.1 percent by volume as a mobile phase B, and the gradient elution is carried out, wherein the flow rate is 1mL/min, and the column temperature is 35 ℃; the gradient elution was as follows:
detecting aflatoxin G2, G1, B2, B1: excitation wavelength is 365nm and emission wavelength is 450nm for 0-20 min; detecting zearalenone: 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm;
detection of ochratoxin a: 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm.
2. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven as claimed in claim 1, wherein the method further comprises the following steps:
precisely measuring a proper amount of reference substance, adding methanol for dilution to obtain the reference substance, wherein the sample volume of the reference substance is 2, 5, 10, 15 and 20 mu l, injecting the reference substance into a liquid chromatograph, recording peak areas, taking the concentration (X, mu g and mL) of each reference substance as a horizontal coordinate, taking a peak area Y as a vertical coordinate, and obtaining the regression equation of each component as follows:
3. the method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven according to claim 2, wherein the preparation method of the aflatoxin reference substance is as follows: control solution lot No. 610001-202107, michem Acad, B 1 1.08μg/mL、B 2 Concentration 0.33. Mu.g/mL, G 1 The concentration is 1.01 mu G/mL, G 2 Precisely measuring 1mL of reference solution with the concentration of 0.33 mug/mL, placing the reference solution into a 20mL measuring flask, and diluting the reference solution to a scale with methanol to obtain a stock solution; precisely measuring 0.4ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
4. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven according to claim 2, wherein the preparation method of zearalenone reference substance is as follows: the control solution is prepared by the steps of (1) performing accurate measurement on a control solution batch No. 610022-202101, a middle hospital, 10.1 mu g/ml, putting 1ml of the control solution into a 10ml measuring flask, and diluting the control solution to a scale with methanol to obtain a stock solution; precisely measuring 1ml of the stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking.
5. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven as claimed in claim 2, wherein the ochratoxin a reference substance is prepared by the following steps: the batch number of the reference substance solution 2B00G12, purapang, 100.9 mu G/ml, precisely measuring 0.1ml of the reference substance solution, placing the reference substance solution in a 100ml measuring flask, and diluting the reference substance solution to a scale with methanol to obtain a stock solution; precisely measuring 2.5ml of the stock solution, placing into a 100ml measuring flask, diluting with methanol to scale, and shaking.
6. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven as claimed in claim 1, which is characterized by further comprising the following steps:
taking one part of the mixed standard solution of the reference substance, injecting samples for 0h, 4h, 8h, 12h, 16h, 20h and 24h respectively, wherein the injection amount is 20 mu l, measuring the peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A respectively and calculating RSD, and the results are as follows:
。
7. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven as claimed in claim 1, which is characterized by further comprising the following steps:
taking 5.0G of medicated leaven powder, precisely weighing 3 parts, respectively placing into 500mL conical flasks with stoppers, respectively adding 0.8mL of aflatoxin reference stock solution, 1mL of zearalenone reference stock solution and 0.2mL of ochratoxin reference stock solution according to the method operation of claim 1, injecting 10 μ L of sample for determination, and calculating the recovery rate, wherein the average sample addition recovery rates (n = 6) of G2, G1, B2, B1, zearalenone and ochratoxin A are respectively as follows: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD of 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
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