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national standards of People's Republic of China
GB/T 18979-2003
Determination of aflatoxin in foods by immunoaffinity chromatography and high performance liquid chromatography
De te rm in at io na fla to xins content
Cleanup by immunoaffinity chromatogaphy
In foodand
Determination by
High-performance liquid chromatigraphy and fluorometer
2003-02-21 released 2003-08-01 implementation of Xiongjiajia k Wi A * 1 M released
GB/T 18979-2003
The standard of Yueming Qing was proposed by the Beijing Municipal Bureau of Quality and Technical Supervision.
This standard is under the jurisdiction of the National Food Industry Standardization Technical Committee.
This standard was drafted: Beijing Product Quality Supervision and Inspection Institute, Qingdao Exit Inspection and Quarantine Bureau, National Standard Materials Research Center.
The main drafters of this standard: Wang Jing, Zhang Peng, Zhang Yibing, Shao Mingwu.
This standard is the first release.
GB/T 18979-2003
Determination of aflatoxin in foods Immunoaffinity chromatography purification High performance liquid chromatography and spectrophotometric range This standard specifies immunoaffinity chromatography purification, high performance liquid chromatography and immunoaffinity chromatography purification-fluorescence spectrophotometry The conditions and detailed analysis steps of aflatoxins in foods were determined.
This standard applies to the determination of aflatoxins in corn, peanuts and their products (peanut butter, peanut kernels, peanuts), rice, wheat, vegetable oils, soy sauce, vinegar and other foods.
Detection limit of aflatoxin in the sample: immunoaffinity chromatography purification, high performance liquid chromatography for the determination of aflatoxin B; and aflatoxin B, 玖, G,, G. The total detection limit is 1k g/kg. Fluorescence spectrophotometric determination of aflatoxin B by immunoaffinity chromatography
The total detection limit of G, G is 1m g/kg, and the detection limit of soy sauce samples is 2.5 pg/kg.
2 immunoaffinity chromatography purification high performance liquid chromatography
2.1 Method summary The sample is extracted with methanol-water. After the extract is filtered and diluted, the filtrate is purified by immunoaffinity chromatography containing aflatoxin-specific antibody. The antibody has specificity for aflatoxin BBz, GG: Aflatoxin is cross-linked to antibodies in the chromatographic medium. The impurities on the immunoaffinity column were removed with water or Tween-20/PBS, eluted with methanol through an immunoaffinity column, and the eluate was derivatized by a high performance liquid chromatography column with a fluorescence detector followed by iodine solution. The content of aspergillus.
2.2 Reagents and solutions Unless otherwise specified, only analytical reagents, double distilled water, are used.
2.2.1 Methanol (CH, OH): chromatographically pure.
2.2.2 Methanol-water (7+3): Take 70 mL of methanol and add 30 mL of water.
2.2.3 Methanol-water (8+2): Take 80 mL of methanol plus 20 mL of water.
2.2.4 Methanol-water (45+55): Take 45 mL of methanol plus 55 mL of water.
2.2.5 Benzene: chromatographically pure.
2.2.6 Ethyl acetate: pure chromatographic.
2.2.7 Benzene-acetonitrile (98+2): Take 2 mL of acetonitrile and add 98 mL of benzene.
2.2.8 Sodium (NaCI),
2.2.9 Disodium hydrogen phosphate (NaiH PO,),
2.2. 10 potassium dihydrogen phosphate (KHzP04).
2.2. 11 Potassium chloride (KCl) e
2.2 .1 2 PBS buffer solution: Weigh 8.0 g of sodium chloride, 1.2 g of disodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate, 0.2 g of potassium, use
Dissolve in 990 mL of pure water, then adjust the pH to 7.0 with concentrated hydrochloric acid, and finally dilute to 1 000 ML with pure water.
2.2.1 3 Tween-20/PBS solution (0.1 00): Take 1m l-Tween-20, add PBS buffer solution and dilute to 100 00m L,
2.2.1 4 pH7.0 phosphate buffer solution: Mix 25.0 m L 0.2 m ol/L potassium dihydrogen phosphate solution with 29.1 m L 0.1 m ol/L sodium hydroxide solution, and dilute to 100 mL.
GB/T 18979- 2003
2.2. 15 Aflatoxin Standard (Aflatoxin BB2, GG): Purity) 9900
2-2. 16 Aflatoxin standard stock solution: Prepared separately with benzene-acetonitrile (98+2) solution. 100 mg/mL aflatoxin B, ,
B2, G,, G: Standard stock solution, stored at 4 ° C for use.
2.2. 17 Aflatoxin mixed standard working solution: accurately transfer appropriate amount of aflatoxin BB 2, GG: standard stock solution, diluted with benzene-acetonitrile (98+2) solution into a mixed standard working solution.
2.2. 18 post-column derivatization solution (0.05% iodine solution): weighed. 1 g of iodine, dissolved in 20 mL of methanol, add pure water to a volume of 200 mL,
Filter with a nylon filter of 0. 45 jam and store at 4 ° C in the dark.
2.3 Instruments and equipment Laboratory routine instruments, equipment and the following.
2.3. 1 High-speed homogenizer: 18 000 r/min-22 000 r/mine
2.3.2 Aflatoxin immunoaffinity column.
2.3.3 glass fiber filter paper: diameter 11 cm, aperture 1. 5 pme
2.3.4 Glass syringe: 10 mL, 20 mLa
2.3.5 Glass test tube: 12 mm in diameter and 75 mm in length, no fluorescence characteristics.
2.3.6 High Performance Liquid Chromatograph: A fluorescence detector with an excitation wavelength of 360 nm and an emission wavelength greater than 420 nm.
2.3.7 Air pressure pump.
2.3.8 Microinjector: 100 KLe
2.3.9 Column: C, column (column length 150 mm, inner diameter 4.6 mm, packing diameter 5 pm) e
2.4 Analysis steps
2.4. 1 extraction
2.4 g。 Samples of rice, corn, wheat, peanuts and their products: accurately weighed (greater than 2 mm) sample 25. 0 g
In a 250 mL stoppered conical flask, add 5.0 g of sodium chloride and methanol-water (7+3) to 125.0 mL (V,), and stir at high speed with a homogenizer.
2 min. Quantitative filter paper filtration, accurately transfer 15.0 mL (V2) filtrate and add 30.0 mL (V,) water to dilute, filter with glass fiber filter paper
1-2 times, until the filtrate is clarified and used.
2.6.1.2 Vegetable oil: accurately weigh 25.0 g of the sample in a 250 m L stoppered conical flask, add 5.0 g of sodium chloride and add methanol to water.
(7+3) to 125.0 m L (V,), and extract 2 m in with high speed stirring by a homogenizer. Quantitative filter paper filtration, accurately remove 15.0 M l-(V2) filtrate and add 30.0 mL (ji) water to dilute, filter 1-2 times with glass fiber filter paper, until the filtrate is clarified and set aside.
2.4.1.3 Soy sauce: Accurately weigh 50.0 g of sample in 250.0 mL stoppered conical flask, add 2.5 g of sodium chloride and add methanol-water
(8 12 2) to 100.0 mL (V4), use a homogenizer to extract 1 mine quantitative filter paper by high-speed stirring, accurately remove 10. 0 mL (V) filtrate and add 40.0 mL (V,) water to dilute with glass. The fiber filter paper was filtered 1-2 times until the filtrate was clarified and used.
2.4.1.4 Vinegar: Accurately weigh 5.0 g of sample, add 1.0 g of sodium chloride, and dilute to 25.0 m L with pH 7.0 phosphate buffer solution.
(V,), mix, quantitative filter paper filtration. Take 10.0 nt L (ji) filtrate and add 10.0 m L (recognition) buffer, mix and filter 1-2 times with glass fiber filter paper until the filtrate is clarified and set aside.
2.4.2 Purification
2.4.2. 1 Rice, corn, wheat, peanuts and their products, vegetable oils and fats: the immunoaffinity column was connected under a 20. 0 ml glass syringe.
Accurately transfer 15.0 mL (V4) sample extract into a glass syringe, connect the air pressure pump to the glass syringe, adjust the pressure so that the solution slowly passes through the immunoaffinity column at a flow rate of about 6 mI./min until 2 mL-3 The mL air passes through the column. The column was rinsed twice with 10 mL of water, all of the effluent was discarded, and 2 mL - 3 mL of air was passed through the column. Accurately add 1.0 mL (V) chromatographic grade methanol elution at a flow rate of 1 mL/min to 2 mL/min. Collect all eluates in glass tubes for testing.
2.4.2.2 Soy sauce: Attach the immunoaffinity column to a 10.0 m L glass syringe. Accurately remove 10.0 mL of the soy sauce sample extract into the glass syringe, connect the air pressure pump to the glass syringe, and adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 6 ml-/min. Wash with 10 mL of 0.1 tweet Tween-20/PBS, rinse the column twice with 10 m1_water, discard all effluent, and pass 2 mL-3 mL of air through the column. Accurately add 1.0 mL
All eluates were collected in glass tubes for testing.
2.4.2.3 Vinegar: Attach the immunoaffinity column to a 10.0 m L glass syringe. Accurately remove 10.0 mL (V,) vinegar sample extract into the glass syringe, connect the air pressure pump to the glass syringe, adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 6 ml-/min, with 10 Wash the Tween-20/PBS solution written in mL 0. 1 and wash the column twice with 10 mL of water, discard all the effluent.
Allow 2 mL-3 mL of air to pass through the column. Accurately add 1.0 mL (V) chromatographic grade methanol elution at a flow rate of 1 mL/min-
At 2 mL/min, collect all the eluate in a glass test tube for testing.
2.4.3 Determination
2.4.3. 1 High Performance Liquid Chromatography Condition Mobile Phase: Methanol-Water (45+55)
Flow rate: . 8M L/min
Post-column derivatization system derivatization solution: .05% iodine solution derivatization solution flow rate: 0.2 m L/min
Reaction tube temperature: 700C
Reaction time: 1m in
2.4.3.2 Quantitatively draw 100p L of aflatoxin mixed standard working solution (2.2.17) into the high performance liquid chromatograph, and determine the response value (peak height or peak area) of the standard solution under the above chromatographic conditions. A high performance liquid chromatogram of aflatoxin BB2, G, and G: standard solutions was obtained.
The reference spectrum is shown in Figure 1.
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Figure 1 Standard spectrum of aflatoxin B, , B 2, GG 2 Take sample eluent 1.0 m L plus human distilled water to a volume of 2.0 m L, and use a sampler to draw 100 p L of high performance liquid chromatography ,
The response value (peak height or peak area) of the sample was measured under the above chromatographic conditions. The concentration of aflatoxin B, B2, G, and G: in the sample is obtained by comparing the response value with the aflatoxin standard solution spectrum:
2.4.4 Blank test water should be used instead of the sample, and the blank test should be carried out according to the procedure of 2.4.1-2.4.3.
2.4.5 Results Calculate the content of aflatoxin BB2, G 1 or G: in the sample (X,) expressed in micrograms per kilogram, calculated according to formula (1):
(c: one ca)·V
W
(1)
GB/T 18979- 2003
among them:
w one department X (v open 3) XV 4 ,....... . .·....···. . (2)
In the formula:
X,— the content of aflatoxin B, , G, or G: in the sample, in micrograms per kilogram of gigagrams/kg);
c,— The content of aflatoxin BB, G, or G in the sample, in micrograms per liter (pg/L);
'. - blank test aflatoxin BB2, G, or G: content, in micrograms per liter (f'g / L);
V—the volume of the final methanol eluent in milliliters (mL);
W—the mass of the sample contained in the final purification eluent, in grams [9);
, the value of the mass of the sample, in grams (g);
V, — total volume of sample and extract in milliliters (mL);
Volume of sample filtrate for V2 dilution, in milliliters (mL);
V3 dilution volume in milliliters (mL);
The volume of the sample extract from the affinity column in milliliters (mL).
The total amount of aflatoxin is the sum of the concentrations of BB2, G, and G2, ie, B, + B2+ G, + G2,
The result of the calculation is expressed as two decimal places.
Immunoaffinity chromatography purification fluorescence spectrophotometry
3.1 Method summary The sample is extracted with methanol-water. After the extract is filtered and diluted, the filtrate is purified by immunoaffinity chromatography containing aflatoxin-specific antibody. This antibody has specificity for aflatoxin B B2, GG: Aflatoxin is cross-linked to antibodies in the chromatographic medium. The impurities on the immunoaffinity column were removed with water. The methanol was eluted by immunoaffinity chromatography column and derivatized with a solution to increase the sensitivity of the assay. The eluate was measured by a fluorophotometer for the total amount of aflatoxin (B, + 玖 + G, ten G 2 ).
3.2 Reagents and solutions Unless otherwise specified, only analytical reagents, double distilled water
3.2.1 Methanol (CH30H): Chromatographically pure.
3.2.2 Methanol-water (7+3): Take 70 mL of methanol and add 30 mL of water.
3.2.3 A drunken water (8+2): Take 80 mL of methanol plus 20 mL of water.
3.2.4 Sodium Sodium (NaCD,
3.2.5 Disodium hydrogen phosphate (NatH PO,).
3.2.6 Potassium dihydrogen phosphate (KH2P O,) o
12. 7 Potassium chloride (KCl)
3.2.8 Australian solution stock solution (0.0 1%): Weigh the appropriate amount of Australian, dissolved in water, formulated into a 0.01% stock solution, 40C stored in the dark.
3.2.9 working solution of Australian solution (0.002 write): Take 10m L0 .0 1% of the Australian solution plus 40m L of water and mix, store in a brown bottle for later use.
It needs to be prepared before each use.
3.2.1 0 quinine sulfate dihydrate (C, oH z, N zO z·H2SO4, 2Hz0),
3.2.1 1 Sulfuric acid solution (0.0 5m ot / L): Take 2.8 m L of concentrated sulfuric acid, slowly add appropriate amount of water, cool down to a volume of 100 00 m L.
3.2.12 Fluorescence photometer calibration solution: Weigh 3.40 g of quinine sulfate (CzoH z,N ,0 2·HzS04·2H20) and dilute to 100 mL with 0.0 5m ot/L sulfuric acid solution. The fluorometer reading of this solution is equivalent. 20 has/L aflatoxin standard solution.
3. 3 Instruments and equipment Laboratory routine instruments, equipment and the following.
3.3. 1 Fluorescence photometer.
3.3.2 sorghum homogenizer. 18 000 r/min--22 000 r/min
GB/T 18979-2003
3.3.3 Aflatoxin toxin immunoaffinity column.
3.3.4 Glass fiber filter paper: diameter 11 cm, aperture 1.5 [am.
3.3.5 Glass syringe: 10 mL, 20 mI.
3.3.6 Glass test tube: 12 mm in diameter and 75 mm in length, no fluorescence characteristics.
3.3.7 Air pressure pump.
3,4 analysis steps
3.4. 1 extract the same as 2.4.1,
3.4.2 Purification is the same as 2. 4. 2,
3.4.3 Determination
3.4.3. 1 The fluorometer is calibrated at an excitation wavelength of 360r an and an emission wavelength of 450n. The .05m ol/L sulfuric acid solution is blank and the reading value of the fluorometer is adjusted. . . . Fag/L; adjust the fluorescence photometer reading value to 20.0 F ag/L with a fluorophotometer calibration solution (3.2.12).
3.4.3.2 Determination of sample solution Take the above purified methanol eluate and add 1.0 m L0 .0 020 o of Oau solution, mix, stand for 1 m in, operate according to the conditions of 3.4.3.1, read in the fluorometer The concentration of aflatoxin (B, + B2+ G, + G2) in the sample. (Kg/L)e
3.4.4 Blank test water should be used instead of the sample, and the blank test should be carried out according to the procedure of 3.4.1--3.4.3.
3.4.5 Results The content of aflatoxin (B, + B2+ G, + G2) in the sample (X2) is expressed in micrograms per kilogram, calculated according to formula (3):
(c: one CD)·V
W
(3)
among them:
W-V, XV 2
(V2 + V,)
XV, (4)
In the formula:
X2—the content of aflatoxin (B, + residual ten G ten G2) in the sample, in micrograms per kilogram of gigagrams/kg)
C2—the content of aflatoxin (B, ten Bz + G, ten G) in the sample, in micrograms per liter (FHB/L);
'. - blank test aflatoxin (B, + B2+ G, + G2) in micrograms per liter of B/L);
V—the volume of the final methanol eluent in milliliters (mL);
W—the mass of the sample contained in the final purification eluent in grams (9);
. – the value of the mass of the sample, in grams (9);
V, — total volume of sample and extract in milliliters (ML);
V—the volume of the sample filtrate for dilution, in milliliters (mL);
V diluent volume in milliliters (mL);
V, — volume of sample extract through the affinity column in milliliters (mL) o
The result of the calculation is expressed as one decimal place.
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ICS 67.050 GB/T 18979-2003