CN104914196A - Determining method of aflatoxin in food - Google Patents
Determining method of aflatoxin in food Download PDFInfo
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- CN104914196A CN104914196A CN201510344861.6A CN201510344861A CN104914196A CN 104914196 A CN104914196 A CN 104914196A CN 201510344861 A CN201510344861 A CN 201510344861A CN 104914196 A CN104914196 A CN 104914196A
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Abstract
The invention discloses a determining method of aflatoxin in food, which has the advantages of low detection cost and short detection time. According to the technical key points, the determining method comprises the steps of extracting and purifying a test substance, precisely weighing and crushing 2.0g of homogenized sample, placing the sample into a 50ml centrifuge tube, adding 4ml of acetonitrile, spirally mixing for 4min, centrifuging for 4min at 4000r/min, precisely measuring 1ml of supernatant, placing the supernatant into a premixing bullet purifying tube containing 200mg HC-C18+25g PSA, spirally mixing for 3min, centrifuging for 10min at super-high speed being 15000r/min, filtering the supernatant through a 0.2 microns of filter member, placing the filtered supernatant into a liquid phase sample feeding bottle. 2 microlieters of extracted and purified sample enters a high performance liquid chromatograph instrument-photochemical derivatization instrument and a fluorescence detector to be detected. The determining method belongs to the technical field of medicine detection.
Description
Technical field
The present invention relates to a kind of assay method of aflatoxin, specifically, relate to the assay method of aflatoxin in food, belong to medical detection technique field.
Background technology
Aflatoxin (AFT) is the compound that a class chemical constitution is similar, is the derivant of dihydrofuran cumarin.Aflatoxin is the secondary metabolite produced primarily of aspergillus flavus aspergillus parasiticus (a.parasiticus), occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.
They are present in soil, animals and plants, various nut, particularly easily pollute the grain oil products such as peanut, corn, rice, soybean, wheat, are mycotoxicosis class mycotoxins that is maximum, that very give prominence to human health risk.
Method in current GB GB/T 18979-2003:
1, extract:
(1) rice, corn, wheat, peanut and goods thereof: accurately take sample 25.0g through levigate (granularity is less than 2mm) in 250mL tool plug conical flask, add 5.0g sodium chloride and methanol-water (7+3) to 125.0mL, extract 2min with homogenizer high-speed stirred.Quantitative filter paper filters, and accurately pipettes 15.0mL filtrate and adds the dilution of 30.0mL water, filtering 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(2) vegetable fat: accurately take sample 25.0g in 250mL tool plug conical flask, adds 5.0g sodium chloride and adds methanol-water (7+3) to 125.0mL, extracts 2min with homogenizer high-speed stirred.Quantitative filter paper filters, and accurately pipettes 15.0mL filtrate and adds the dilution of 30.0mL water, filtering 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(3) soy sauce: accurately take sample 50.0g in 250.0mL tool plug conical flask, add 2.5g sodium chloride and add methanol-water (8+2) to 100.0mL, extract 1min quantitative filter paper with homogenizer high-speed stirred to filter, accurately pipette 10.0mL filtrate and add the dilution of people 40.0mL water, filter 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(4) vinegar: accurately take 5.0g sample, adds 1.0g sodium chloride, is diluted to 25.0mL with pH7.0 phosphate buffered solution, mixing, and quantitative filter paper filters.Get 10.0mL filtrate and add 10.0mL damping fluid, mixing, filter 1-2 time with glass fiber filter paper, to filtrate clarification, for subsequent use.
2, purify
(1) rice, corn, wheat, peanut and goods thereof, vegetable fat: under immune affinity column being connected to 20.0mL glass syringe.Accurately pipette in 15.0mL sample extracting solution implantation glass syringe, air pressure pump is connected with glass syringe, regulate pressure to make solution slow transit through immune affinity column with about 6mL/min flow velocity, until 2mL-3mL air passes through cylinder.With 10mL water wash pillar twice, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.
(2) soy sauce: under immune affinity column being connected to 10.0mL glass syringe.Accurately pipette in 10.0mL soy sample extract implantation glass syringe, air pressure pump is connected with glass syringe, regulate pressure to make solution slow transit through immune affinity column with about 6ml-/min flow velocity.With the tween-20/PBS cleaning of 10mL 0.1%, then with 10m1 water cleaning pillar twice, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add people 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.
(3) vinegar: under immune affinity column being connected to 10.0mL glass syringe.Accurately pipette in 10.0mL vinegar sample extracting solution implantation glass syringe, air pressure pump is connected with glass syringe, pressure is regulated to make solution slow transit through immune affinity column with about 6mL-/min flow velocity, with the Tween-20/PBS solution cleaning of 10mL 0.1%, pillar is cleaned twice again with 10mL water, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.
3. high-efficient liquid phase chromatogram condition: mobile phase: methanol-water (45+55); Flow velocity: 0.8mL/min; Post column derivatization system: derivative solution: 0.05% iodine solution; Derivative solution flow velocity: 0.2mL/min; Reaction tube temperature: 70 DEG C; Reaction time: 1min.
Adopt the method GB to do a collection of inspection product experimental cost of experiment and about need 160 yuan, experiment pre-treatment about 1.5h consuming time completes a collection of inspection product, and does not just consider the flushing of chromatographic column with isocratic elution.
Summary of the invention
For the problems referred to above, the object of the invention is to, provide a kind of testing cost low, the assay method of aflatoxin in the food that detection time is short.
In food, an assay method for aflatoxin, is characterized in that, comprises the steps: successively
1) test article extraction and cleaning
A) for rice, corn, wheat, peanut and goods extraction and purification methods thereof be: precision takes the sample 2.0g after pulverizing homogeneous, put in 50ml centrifuge tube, add acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put son containing in 200mgHC-C18+25mg PSA premixed bullet purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product.
B) for soy sauce, vinegar, vegetable fat extraction and purification methods be: precision takes the sample 2.0g mixed, put in 50ml centrifuge tube, precision adds acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put son containing in 200mgHC-C18+25mg PSA premixed bullet purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product.
3) by step 1) in extraction and cleaning after sample get respectively 2 μ l enter high performance liquid chromatograph-photochemical derivatization instrument add fluorescence detector detect;
Described chromatogram phase condition is
Instrument: Agilent 1290 high performance liquid chromatographs-photochemical derivatization instrument adds fluorescence detector;
Chromatographic column: ZORBAX Eclipse Plus C18 3.5um 4.6 × 100mm;
Flow velocity: 1ml/ml;
Column temperature: 40 DEG C;
Sample size: 2 μ l;
Detecting device: excitation wavelength is 360nm, emission wavelength is 450nm
Mobile phase: methanol-water 50:50.
Compared with prior art, technical scheme provided by the invention has following technological merit:
1) technical scheme testing cost provided by the invention is lower, and testing cost is 35 yuan, does a collection of inspection product experimental cost of experiment about need 160 yuan by GB;
2) technical scheme provided by the invention is short for detection time, completes a collection of detection product and only needs 35min, by GB experiment pre-treatment about 1.5h consuming time.
3) method provided by the invention adds the flushing of organic phase at high proportion after detected material has gone out peak, can the oily cleaning down in chromatographic column, and the method that GB provides then just does not consider the flushing of chromatographic column with isocratic elution.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention 1 supplying method detects chromatogram;
Fig. 2 is that the method that GB provides detects chromatogram.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not form any limitation of the invention, and the amendment of anyone limited number of time made in right of the present invention is still in right of the present invention.
Embodiment 1
1, extraction and cleaning
Rice, corn, wheat, peanut and goods thereof: precision takes the sample 2.0g after pulverizing homogeneous, put in 50ml centrifuge tube, add acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put (200mgHC-C18+25mg PSA premixed) in sub warhead purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product.
Soy sauce, vinegar, vegetable fat: precision takes the sample 2.0g mixed, put in 50ml centrifuge tube, precision adds acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put (200mgHC-C18+25mg PSA premixed) in sub warhead purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product.
2, liquid-phase condition:
Instrument: Agilent 1290 high performance liquid chromatographs-photochemical derivatization instrument adds fluorescence detector;
Chromatographic column: ZORBAX Eclipse Plus C18 3.5um 4.6 × 100mm;
Flow velocity: 1ml/ml;
Column temperature: 40 DEG C
Sample size: 2 μ l;
Detecting device: excitation wavelength is 360nm, emission wavelength is 450nm mobile phase: methanol-water; Chromatogram consults Fig. 1, and peak is AFG 2, G1, B2, B1 from left to right successively
Mobile phase consults table 1:
Table 1
Spectral data consults table 2.
Table 2
National standard method:
1, extract:
(1) rice, corn, wheat, peanut and goods thereof: accurately take sample 25.0g through levigate (granularity is less than 2mm) in 250mL tool plug conical flask, add 5.0g sodium chloride and methanol-water (7+3) to 125.0mL, extract 2min with homogenizer high-speed stirred.Quantitative filter paper filters, and accurately pipettes 15.0mL filtrate and adds the dilution of 30.0mL water, filtering 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(2) vegetable fat: accurately take sample 25.0g in 250mL tool plug conical flask, adds 5.0g sodium chloride and adds methanol-water (7+3) to 125.0mL, extracts 2min with homogenizer high-speed stirred.Quantitative filter paper filters, and accurately pipettes 15.0mL filtrate and adds the dilution of 30.0mL water, filtering 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(3) soy sauce: accurately take sample 50.0g in 250.0mL tool plug conical flask, add 2.5g sodium chloride and add methanol-water (8+2) to 100.0mL, extract 1min quantitative filter paper with homogenizer high-speed stirred to filter, accurately pipette 10.0mL filtrate and add the dilution of people 40.0mL water, filter 1-2 time by glass fiber filter paper, to filtrate clarification, for subsequent use.
(4) vinegar: accurately take 5.0g sample, adds 1.0g sodium chloride, is diluted to 25.0mL with pH7.0 phosphate buffered solution, mixing, and quantitative filter paper filters.Get 10.0mL filtrate and add 10.0mL damping fluid, mixing, filter 1-2 time with glass fiber filter paper, to filtrate clarification, for subsequent use.
2, purify:
(1) rice, corn, wheat, peanut and goods thereof, vegetable fat: under immune affinity column being connected to 20.0mL glass syringe.Accurately pipette in 15.0mL sample extracting solution implantation glass syringe, air pressure pump is connected with glass syringe, regulate pressure to make solution slow transit through immune affinity column with about 6mL/min flow velocity, until 2mL-3mL air passes through cylinder.With 10mL water wash pillar twice, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.
(2) soy sauce: under immune affinity column being connected to 10.0mL glass syringe.Accurately pipette in 10.0mL soy sample extract implantation glass syringe, air pressure pump is connected with glass syringe, regulate pressure to make solution slow transit through immune affinity column with about 6ml-/min flow velocity.With the tween-20/PBS cleaning of 10mL 0.1%, then with 10m1 water cleaning pillar twice, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add people 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.
(3) vinegar: under immune affinity column being connected to 10.0mL glass syringe, accurately pipette in 10.0mL vinegar sample extracting solution implantation glass syringe, air pressure pump is connected with glass syringe, pressure is regulated to make solution slow transit through immune affinity column with about 6mL-/min flow velocity, with the Tween-20/PBS solution cleaning of 10mL 0.1%, again with 10mL water cleaning pillar twice, discard whole efflux, and make 2mL-3mL air pass through cylinder.Accurately add 1.0mL hplc grade methanol wash-out, flow velocity is 1mL/min-2mL/min, collects whole eluent in glass test tube, for detection.High-efficient liquid phase chromatogram condition: mobile phase: methanol-water (45+55); Flow velocity: 0.8mL/min; Post column derivatization system; Derivative solution: 0.05% iodine solution; Derivative solution flow velocity: 0.2mL/min; Reaction tube temperature: 70 DEG C; Reaction time: 1min; Chromatogram consults 2.
Claims (1)
1. the assay method of aflatoxin in food, is characterized in that, comprise the steps: successively
1) test article extraction and cleaning
A) for rice, corn, wheat, peanut and goods extraction and purification methods thereof be: precision takes the sample 2.0g after pulverizing homogeneous, put in 50ml centrifuge tube, add acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put son containing in 200mgHC-C18+25mg PSA premixed bullet purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product;
B) for soy sauce, vinegar, vegetable fat extraction and purification methods be: precision takes the sample 2.0g mixed, put in 50ml centrifuge tube, precision adds acetonitrile 4ml, vortex mixing 4min, centrifugal 4min under 4000 turns/min, precision measures supernatant 1ml, put son containing in 200mgHC-C18+25mg PSA premixed bullet purification pipe, vortex mixing 3min, 15000 turns/min ultracentrifugation 10min, get supernatant to cross 0.2 micron membrane filter and put in liquid-phase inlet bottle, to obtain final product;
2) by step 1) in extraction and cleaning after sample get respectively 2 μ l enter high performance liquid chromatograph-photochemical derivatization instrument add fluorescence detector detect;
Described chromatogram phase condition is
Instrument: Agilent 1290 high performance liquid chromatographs-photochemical derivatization instrument adds fluorescence detector;
Chromatographic column: ZORBAX Eclipse Plus C18 3.5um 4.6 × 100mm;
Flow velocity: 1ml/ml;
Column temperature: 40 DEG C;
Sample size: 2 μ l;
Detecting device: excitation wavelength is 360nm, emission wavelength is 450nm
Mobile phase: methanol-water 50: 50.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108732260A (en) * | 2018-02-28 | 2018-11-02 | 中国林业科学研究院亚热带林业研究所 | The extraction and purification methods of aflatoxin B1 in one vegetable oil |
| CN110108822A (en) * | 2019-05-14 | 2019-08-09 | 苏州贞成分析仪器有限公司 | Aflatoxin sample extraction detection method and its purification pipette tips in a kind of edible vegetable oil |
| CN110108811A (en) * | 2019-05-20 | 2019-08-09 | 青岛贞正分析仪器有限公司 | Aflatoxin sample purification detection method and its purification pipette tips in a kind of brewing seasoning |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732260A (en) * | 2018-02-28 | 2018-11-02 | 中国林业科学研究院亚热带林业研究所 | The extraction and purification methods of aflatoxin B1 in one vegetable oil |
| CN110108822A (en) * | 2019-05-14 | 2019-08-09 | 苏州贞成分析仪器有限公司 | Aflatoxin sample extraction detection method and its purification pipette tips in a kind of edible vegetable oil |
| CN110108811A (en) * | 2019-05-20 | 2019-08-09 | 青岛贞正分析仪器有限公司 | Aflatoxin sample purification detection method and its purification pipette tips in a kind of brewing seasoning |
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Application publication date: 20150916 |