CN115452990B - 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 50
- 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 49
- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 37
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 34
- 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 30
- 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 30
- 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 30
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 title claims abstract description 29
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 36
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 21
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- 238000010828 elution Methods 0.000 claims abstract description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 6
- 229930166256 Aflatoxin G2 Natural products 0.000 claims abstract description 5
- WPCVRWVBBXIRMA-WNWIJWBNSA-N Aflatoxin G2 Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1[C@@H]1CCO[C@@H]1O2 WPCVRWVBBXIRMA-WNWIJWBNSA-N 0.000 claims abstract description 5
- 239000002100 aflatoxin G2 Substances 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 96
- 239000013558 reference substance Substances 0.000 claims description 25
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- 229930132918 Aflatoxin B2 Natural products 0.000 claims description 2
- 229930063498 Aflatoxin G1 Natural products 0.000 claims description 2
- XWIYFDMXXLINPU-WNWIJWBNSA-N Aflatoxin G1 Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1[C@@H]1C=CO[C@@H]1O2 XWIYFDMXXLINPU-WNWIJWBNSA-N 0.000 claims description 2
- 239000002115 aflatoxin B1 Substances 0.000 claims description 2
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 2
- 239000002097 aflatoxin B2 Substances 0.000 claims description 2
- WWSYXEZEXMQWHT-WNWIJWBNSA-N aflatoxin B2 Chemical compound C=1([C@@H]2CCO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O WWSYXEZEXMQWHT-WNWIJWBNSA-N 0.000 claims description 2
- 239000002098 aflatoxin G1 Substances 0.000 claims description 2
- 229930020125 aflatoxin-B1 Natural products 0.000 claims description 2
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- QRARGUIFAGCOOA-UHFFFAOYSA-N aspertoxin Chemical compound O1C2=C(C3(C=COC3O3)O)C3=CC(OC)=C2C(=O)C2=C1C=CC=C2OC QRARGUIFAGCOOA-UHFFFAOYSA-N 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/14—Preparation by elimination of some components
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The invention relates to a method for detecting mycotoxins in foods and medicines, in particular to a method for simultaneously detecting aflatoxins, zearalenone and ochratoxin A in medicated leaven by using an immunoaffinity column purification-post-column photochemical derivatization-high performance liquid chromatography. According to the method, an XBiridge Phenyl column is used, acetonitrile is used as a mobile phase A, 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 (detection of aflatoxin G2, G1, B2, B1); 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm (detection of zearalenone); 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm (detection of ochratoxin A). The method is simple, convenient and quick, has good purification effect, and all technical indexes meet the requirement of mycotoxin detection, and can meet the limit detection requirement that aflatoxin, zearalenone and ochratoxin A in medicated leaven can be detected simultaneously by 1 sample injection.
Description
Technical Field
The invention relates to a method for detecting mycotoxins in foods and medicines, in particular to a method for simultaneously detecting aflatoxins, zearalenone and ochratoxin A in medicated leaven by using an immunoaffinity column purification-post-column photochemical derivatization-high performance liquid chromatography.
Background
Aflatoxins (AFT) are difuranoid toxins produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. There are about 20 kinds of derivatives, named B1, B2, G1, G2, M1, M2, GM, P1, Q1, toxic alcohol, etc. Among them, B1 has the greatest toxicity and the strongest carcinogenicity. After the animal eats the aflatoxin-contaminated feed, trace amounts of toxins can be detected in the liver, kidney, muscle, blood, milk and eggs. Aflatoxins and their producing strains are widely distributed in nature, some strains produce more than one type of aflatoxin, and there are strains in aflatoxins that do not produce any type of aflatoxin. Aflatoxin mainly pollutes grain and oil and products thereof, and various plant and animal foods can also be polluted. The toxin-producing aspergillus is easy to parasitize and produce toxin in cereal crops, oil crop seeds and processing byproducts thereof with higher moisture content (the moisture content is lower than 12 percent and cannot be propagated), so that the toxin-producing aspergillus is mildewed and deteriorated, and people can ingest the food or the processing byproducts thereof by mistake and absorb the toxin into human bodies through the digestive tract to poison.
Zearalenone (Zearalenone), also known as F-2 toxin, was first isolated from corn with scab. Zearalenone mainly pollutes grains such as corn, wheat, rice, barley, millet and oat. The zearalenone has high heat resistance and is completely destroyed after being treated for 1h at 110 ℃. Zearalenone has estrogen-like effect, can cause acute and chronic poisoning of animals, cause abnormal reproduction functions of animals and even death of animals, and can cause huge economic loss for animal farm. Zearalenone is a metabolite of gibberella zeae.
Aflatoxin (aspertoxin), a compound produced by the metabolism of aspergillus flavus (Aspergillus flavus). Is a metabolite or secondary metabolite produced by aspergillus, usually present in mildewed grains, which may cause serious harm such as teratogenicity, mutation and carcinogenesis once entering the human body through the food chain, and thus is of great concern in global food safety. Toxicology studies indicate that ochratoxin a has strong hepatotoxicity and renal toxicity, and has teratogenic, mutagenic and carcinogenic effects. Ochratoxin a is widely found in cereals and cereal products, spices and coffee beans, and is a serious hazard to human health.
Medicated leaven is prepared by adding herba Polygoni Hydropiperis, herba Artemisiae Annuae, semen Armeniacae amarum, etc. into flour or testa Tritici, mixing, and fermenting. Flour or bran is added in the processing process, and mycotoxins such as aflatoxin, zearalenone, ochratoxin A and the like can be brought in. In addition, mycotoxins such as aflatoxin and the like are detected from various traditional Chinese medicinal materials at present. Along with the increasing pollution of mycotoxins to traditional Chinese medicinal materials, it is necessary to establish a method for efficiently and accurately detecting trace amounts of mycotoxins such as aflatoxin, zearalenone, ochratoxin A and the like in medicated leaven, and technical support is provided for the quality of the medicated leaven. However, the existing detection methods for mycotoxins such as aflatoxin, zearalenone, ochratoxin A and the like in foods and medicines mainly comprise thin layer chromatography, enzyme-linked immunosorbent assay, immunoaffinity column purification fluorescence photometry, immunoaffinity column purification high performance liquid chromatography, multifunctional column chromatography purification high performance liquid chromatography, mass spectrometry and the like. The first 3 methods have poor quantitative accuracy, the second 2 methods can only detect single mycotoxins, and the cost requirements and personnel requirements of mass spectrometry on enterprises are high.
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 medicated leaven, which adopts immunoaffinity column purification-post-column photochemical derivatization-high performance liquid chromatography to simultaneously detect various mycotoxins.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven comprises the steps of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin 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 percent concentration, performing ultrasonic treatment for 40min, transferring into a 50ml centrifuge tube, and centrifuging for 10min at 6000 rpm; precisely weighing 10ml of supernatant, diluting with water to scale in a 50ml measuring flask, shaking, centrifuging at 6000 rpm for 10min, weighing 25ml of supernatant, eluting with immunoaffinity column, adding 20ml of water after eluting, eluting again, discarding eluent, squeezing out water in the column, eluting with 2ml of methanol, collecting eluent, shaking;
2) High performance liquid chromatography assay
The equipment adopts Shimadzu high performance liquid chromatograph and photochemical derivative instrument manufactured by Germany LC-Tech company, and usesPhenyl column uses acetonitrile as mobile phase A, acetic acid solution with 0.1 volume percent concentration as mobile phase B, gradient elution is carried out, the flow rate is 1mL/min, and the column temperature is 35 ℃; gradient elution was as follows:
Detection of aflatoxins G2, G1, B2, B1: 0-20 min, excitation wavelength 365nm, emission wavelength 450nm; detection of 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 injection amount 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.mL) of each reference substance as an abscissa and the peak area Y as an ordinate, and the regression equation of each component is as follows:
| composition of the components | 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 control comprises the following steps: control solution batch No. 610001-202107, middle school, B 1 1.08μg/mL、B2 concentration 0.33 μg/mL, G 1 concentration 1.01 μg/mL, G 2 concentration 0.33 μg/mL, precisely measuring control solution 1mL, placing in 20mL measuring flask, diluting with methanol to scale, and collecting stock solution; precisely measuring 0.4ml of stock solution, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.
Preferably, the preparation method of the zearalenone control comprises the following steps: reference substance solution batch No. 610022-202101, middle school, 10.1 mug/ml, precisely measuring 1ml of reference substance solution, placing in a 10ml measuring flask, diluting with methanol to scale, and taking as stock solution; precisely measuring 1ml of stock solution, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.
Preferably, the method for preparing the ochratoxin A reference substance comprises the following steps: control solution lot number 2B00G12, pribon, 100.9 μg/ml, precisely measuring 0.1ml of control solution, placing into a 100ml measuring flask, diluting with methanol to scale, and taking as stock solution; precisely measuring 2.5ml of stock solution, placing in a 100ml measuring flask, diluting to scale with methanol, and shaking.
Preferably, the method further comprises the steps of:
Taking one part of reference substance mixed standard solution, respectively carrying out sample injection at 0, 4h, 8h, 12h, 16h, 20h and 24h, wherein the sample injection amount is 20 μl, respectively measuring peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A, and calculating RSD, and the result is as follows:
Preferably, the method further comprises the steps of:
Taking 5.0G of medicated leaven powder, weighing 3 parts, respectively placing the medicated leaven powder and the medicated leaven powder into 500mL conical flasks with plugs, respectively adding 0.8mL of aflatoxin reference stock solution, 1mL of zearalenone reference stock solution and 0.2mL of ochratoxin reference substance, operating according to the method disclosed by the application, sampling 10 mu L of sample, measuring, calculating recovery rates, and respectively obtaining average sample recovery rates (n=6) of G2, G1, B2, B1, zearalenone and ochratoxin A: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
By adopting the technical scheme, the method adopts immunoaffinity column purification, photochemical derivatization after column and high performance liquid chromatography for simultaneous detection, is simple, convenient and quick, has good purification effect, meets the requirements of mycotoxin detection on all technical indexes, and can meet the limit detection requirement of simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven by 1 sample injection.
Drawings
FIG. 1 is a liquid chromatography of a mixed control of aflatoxin, zearalenone and ochratoxin A.
FIG. 2 is a liquid chromatograph of sample L05.
Detailed Description
1. Test method
1.1 Instruments
The Shimadzu high performance liquid chromatograph, the photochemistry derivatization instrument is manufactured by Germany LC-Tech company; a high-speed refrigerated centrifuge, a constant-temperature ultrasonic cleaner, METTLER TOLEDO XP (parts per million analytical balance), XP204 (parts per million analytical balance); immunoaffinity columns-a variety of toxins (aflatoxin, zearalenone, ochratoxin a) are produced by peninsula prim bioengineering limited.
1.2 Reagents
Acetonitrile, methanol (chromatographic purity); acetic acid (superior purity); aflatoxin (B1, B2, G1, G2) mix control, zearalenone, ochratoxin a control; the water for the experiment was ultrapure water prepared by Millipore.
1.3 Reagents
Medicated leaven is provided by Zhejiang tungjuntang Chinese herbal pieces limited.
1.4 Chromatographic conditions and System adaptations
Chromatographic column: Phenyl column (4.6X250 mm,5 μm) was eluted with a gradient of acetonitrile-0.1% acetic acid solution (30:70) as the mobile phase (see Table below). 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 (detection of aflatoxin G2, G1, B2, B1); 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm (detection of zearalenone); 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm (detection of ochratoxin A).
Sample injection amount of reference substance: 2. 5, 10, 15 and 20 mu l of sample injection amount: 20 μl.
1.5 Preparation of solutions
1.5.1 Control solution
(1) Aflatoxin control (batch No. 610001-202107, middle school, B 1 1.08μg/mL、B2 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 a20 ml measuring flask, and diluted to the scale with methanol to prepare a stock solution. Precisely measuring 0.4ml of stock solution, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.
(2) Zearalenone control (batch No. 610022-202101, middle school of care, 10.1. Mu.g/ml): 1ml of the control solution was precisely measured and placed in a 10ml measuring flask, and diluted to the scale with methanol to prepare a stock solution. Precisely measuring 1ml of stock solution, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.
(3) Ochratoxin A control (lot number 2B00G12, pribon, 100.9. Mu.g/ml), precisely measuring 0.1ml of control solution, placing in a 100ml measuring flask, and diluting to scale with methanol to obtain stock solution. Precisely measuring 2.5ml of stock solution, placing in a 100ml measuring flask, diluting to scale with methanol, and shaking.
1.5.2 Sample solutions
Precisely weighing 5g of the test sample, precisely adding 50ml of 80% methanol, performing ultrasonic treatment for 40min, transferring into a 50ml centrifuge tube, and centrifuging for 10min (6000 revolutions). Precisely weighing 10ml of supernatant, diluting with water to scale in a 50ml measuring flask, shaking, centrifuging for 10min (6000 revolutions), weighing 25ml of supernatant, eluting with immunoaffinity column, adding 20ml of water after eluting, eluting again, discarding eluent, squeezing out water in the column, eluting with 2ml of methanol, collecting eluent, and shaking.
Methodology investigation
2.1 Standard Curve
Precisely measuring a proper amount of reference substance, adding methanol for dilution to obtain reference substance sample injection amount of 2, 5, 10, 15 and 20 μl, injecting into a liquid chromatograph, recording peak area, taking the concentration (X, μg.mL) of each reference substance as abscissa, and peak area Y as ordinate, wherein the regression equation of each component is shown in Table 1.
Table 1 regression equation, correlation coefficient and r (n=6)
| Composition of the components | 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 Stability test of control
Taking one part of the reference substance mixed standard solution, respectively carrying out sample injection at 0, 4h, 8h, 12h, 16h, 20h and 24h, wherein the sample injection amount is 20 μl, respectively measuring the peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A, and calculating RSD, and the results are shown in Table 2.
Table 2 stability test of control
| Composition of the components | RSD％ |
| G2 | 1.48 |
| G1 | 0.85 |
| B2 | 1.82 |
| B1 | 2.38 |
| Zearalenone | 3.19 |
| Ochratoxin A | 1.24 |
2.3 Sample recovery test
Taking 5.0G of medicated leaven powder, weighing 3 parts, placing into 500mL conical flasks with plugs respectively, adding 0.8mL of aflatoxin reference stock solution, 1mL of zearalenone reference stock solution and 0.2mL of ochratoxin reference (the accurate measurement of 0.1mL of reference stock solution and the dilution of 80% methanol to 50mL of scale marks) respectively, operating according to the method of 1.5.2, sampling 10 mu L according to the chromatographic condition of 1.4, measuring, calculating recovery rates, and obtaining the results of G2, G1, B2, B1, zearalenone and ochratoxin A respectively, wherein the average sample recovery rates (n=6) are as follows: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
Third, result
3.1 Sample content determination
The application respectively takes 16 medicated leaven samples produced by 10 companies, respectively prepares test solution according to the method of item 1.5.2, carries out content measurement according to the chromatographic condition of item 1.4, calculates the content of each component in the medicated leaven by adopting an external standard method, and the result is shown in table 3.
TABLE 3 determination of the content of Massa Medicata Fermentata (. Mu.g/kg)
Fourth discussion of
4.1. Extraction method
In a chromatographic column: Phenyl column (4.6X250 mm,5 μm) was eluted with a gradient of acetonitrile-0.1% acetic acid solution (30:70) as the mobile phase (see Table 4 below). 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 (detection of aflatoxin G2, G1, B2, B1); 20.01-25 min, excitation wavelength 274nm, emission wavelength 460nm (detection of zearalenone); 25.01-25 min, excitation wavelength 333nm, and emission wavelength 460nm (detection of ochratoxin A).
TABLE 4 gradient elution procedure
(1) When the extraction solution was changed to 70% acetonitrile, the recovery rates were calculated, and the sample recovery rates of G2, G1, B2, B1, zearalenone, ochratoxin a were respectively: 70.96%, 110.56%, 106.41%, 104.32%, 80.84% and 0.0%.
(2) When the extraction solution was changed to 80% acetonitrile, the recovery rates were calculated, and the sample recovery rates of G2, G1, B2, B1, zearalenone, ochratoxin a were respectively: 96.80%, 114.00%, 111.14%, 103.61%, 97.73% and 0.0%.
4.2. Immunoaffinity column investigation
Consistent with the chromatographic conditions of item "4.1", immunoaffinity column-multiple toxins (aflatoxin, zearalenone, ochratoxin a) were changed to 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 inspection
ZORBAX C18 (4.6X105 mm,5 μm), EXtend C C18 (4.6X105 mm,5 μm), phenomenex C C18 (4.6X105 mm,5 μm),A Phenyl column (4.6X250 mm,5 μm) and other chromatographic columns, and the fact that the 4 components of aflatoxin in the first three columns have drift retention time along with the increase of needle insertion times in the experimental process and finally have partial coincidence, so that the separation degree cannot meet the requirement. And/>Phenyl column (4.6X250 mm,5 μm) remained stable for the duration of the experiment and the peak pattern was good.
4.4. Selection of mobile phase
The comparison of organic phase methanol, acetonitrile, inorganic phase water, 0.1% phosphoric acid, 0.1% acetic acid, 0.2% acetic acid, 0.5% acetic acid, shows that methanol and 0.1% acetic acid solution do not effectively separate the target peak, while acetonitrile and acid solution can separate the target component. The comparison and investigation of the acid solutions with different types and different proportions on the same concentration of the reference substances are studied, the acid concentrations of the different acids and the acid concentration are found to have a certain influence on the peak area, and finally the gradient elution of 0.1% acetic acid and acetonitrile is determined to be selected as a mobile phase, and the comparison result is shown in the table 5 below.
TABLE 5 Peak areas for 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. Various modifications to these embodiments will be readily apparent to those skilled in the art. The generic principles defined herein may be applied to 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 (4)
1. A method for simultaneously detecting aflatoxin, zearalenone and ochratoxin A in medicated leaven comprises the steps of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2; characterized in that the method comprises the following steps:
1) Sample extraction
Precisely weighing 5g of a test sample, precisely adding 50ml of 80% methanol with volume percent concentration, performing ultrasonic treatment for 40min, transferring into a 50ml centrifuge tube, and centrifuging for 10min at 6000 rpm; precisely weighing 10ml of supernatant, diluting with water to scale in a 50ml measuring flask, shaking, centrifuging at 6000 rpm for 10min, weighing 25ml of supernatant, eluting with immunoaffinity column, adding 20ml of water after eluting, eluting again, discarding eluent, squeezing out water in the column, eluting with 2ml of methanol, collecting eluent, shaking;
2) High performance liquid chromatography assay
The measuring equipment adopts an Shimadzu high performance liquid chromatograph and a photochemical derivative instrument produced by Germany LC-Tech company, uses an XBRridge 2 Phenyl column and acetonitrile as a mobile phase A, uses acetic acid solution with 0.1 volume percent concentration as a mobile phase B, carries out gradient elution, and has the flow rate of 1mL/min and the column temperature of 35 ℃; gradient elution was as follows:
Detection of aflatoxins G2, G1, B2, B1: 0. 20 min, excitation wavelength 365nm, emission wavelength 450 nm; detection of zearalenone: 20.01-25 min, excitation wavelength 274 nm and emission wavelength 460nm;
Detection of ochratoxin a: 25.01-25 min, excitation wavelength 333nm, emission wavelength 460nm;
The method further comprises the steps of:
Precisely measuring a proper amount of reference substances, adding methanol for dilution to obtain reference substances with sample injection amounts of 2, 5, 10, 15 and 20 mu l, injecting into a liquid chromatograph, recording peak areas, taking the concentration X of each reference substance, mu g and mL as an abscissa and the peak area Y as an ordinate, and adopting the regression equation of each component as follows:
The method further comprises the steps of:
Taking one part of reference substance mixed standard solution, respectively injecting samples at 0, 4h, 8 h, 12 h, 16h, 20h and 24h, wherein the injection amount is 20 μl, respectively measuring peak area values of G2, G1, B2, B1, zearalenone and ochratoxin A, and calculating RSD, and the result is as follows:
The method further comprises the steps of:
Taking 5.0G of medicated leaven powder, 3 parts of medicated leaven powder, accurately weighing, respectively placing the medicated leaven powder and the medicated leaven powder into 500mL conical flasks with plugs, respectively adding 0.8mL of aflatoxin control stock solution, 1mL of zearalenone control stock solution and 0.2mL of ochratoxin control, performing sample injection by 10 mu L of measurement, calculating recovery rate, and respectively obtaining average sample injection recovery rates of G2, G1, B2, B1, zearalenone and ochratoxin A, wherein n=6: 99.88%, 99.10%, 96.58%, 83.49%, 69.31%, 33.14%, RSD 1.75%, 1.17%, 1.06%, 1.74%, 9.98%, 12.73%, respectively.
2. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven according to claim 1, wherein the preparation method of the aflatoxin control is as follows: reference substance solution batch numbers 610001-202107, middle school, B 1 1.08µg/mL、 B2 with concentration of 0.33 mu G/mL, G 1 with concentration of 1.01 mu G/mL and G 2 with concentration of 0.33 mu G/mL, precisely measuring 1mL of the reference substance solution, placing in a 20mL measuring flask, and diluting to scale with methanol to obtain stock solution; precisely measuring 0.4ml of stock solution, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.
3. The method for simultaneously detecting aflatoxin, zearalenone and ochratoxin a in medicated leaven according to claim 1, wherein the preparation method of the zearalenone control is as follows: reference substance solution batch No. 610022-202101, middle school, 10.1 mug/ml, precisely measuring 1ml of reference substance solution, placing in a 10ml measuring flask, diluting with methanol to scale, and taking as stock solution; precisely measuring 1ml of stock solution, placing in 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 1, wherein the preparation method of the ochratoxin a reference substance is as follows: control solution lot number 2B00G12, pribon, 100.9 μg/ml, precisely measuring 0.1ml of control solution, placing into a 100ml measuring flask, diluting with methanol to scale, and taking as stock solution; precisely measuring 2.5ml of stock solution, placing in a 100ml measuring flask, diluting to scale with methanol, and shaking.
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