State reaches aflatoxin immunoaffinity column dedicated small aflatoxin in foods B1, B2, G1, G2, M1 and M2 detection 1. Brief Introduction: This method is used immunoaffinity column purification, high performance liquid chromatography with fluorescence detection Aflatoxin B1, B2, G1, G2, M1 and M2, this method is applicable to the nuts and the like, dry fruits, cereals, spices, tea, coffee, cocoa, and other food ingredients and feed. Aflatoxin soluble in polar organic reagent, so the use of a uniform matrix aflatoxin methanol / water extract obtained by shaking or dispersion for high fat / oil content of the sample matrix must be added to n-hexane to be skimmed. Then filtered and diluted in PBS. But for the feed, condiments, tea, coffee, cocoa, etc. without Tween solution is diluted with PBS and then filtered through glass fiber filter paper, and then by HPLC post-column derivatization photochemical fluorescence detector. 2, the extraction step instruments, required reagents, solvents aflatoxin immunoaffinity column series: immunoaffinity column series (1ml, 25 / box; 3ml, 15 / box); HPLC apparatus: Agilent 1200 Series contains pump, online degasser, autosampler, fluorescence detector, column oven; photochemical reactor: Photochemical Reactor reaction ring and the other with UV light; Column: XB-C18 5μm, 250 x 4.6 mm; pre-column: C18 4 x 2 mm; homogenizer; horizontal oscillator; pulper; paper; funnel; Erlenmeyer flask: 100 ml; glass fiber filter; SPE adapter and a 50ml capacity injector SPE Adapter10 ml disposable syringes ; analytical balance (precision 0.02 g); 1.8 mL autosampler vial; pipette; SPE device with a vacuum system: SPE Device aflatoxins B1, B2, G1, G2, M1, M2 mixed standard or need to measure single standard; Tween 20; PBS buffer solution: 1.16 g Na2HPO4 / 0.20 g KH2PO4 / 8 g NaCl / 0.2 g KCl dissolved in 900ml water, with HCL or NaOH to adjust PH to 7.4 and then set the volume to 1000ml; disodium hydrogen phosphate; phosphate Potassium dihydrogen; potassium chloride; sodium chloride; deionized water; methanol, AR; methanol, HPLC grade; acetonitrile, HPLC grade; n-hexane, AR; extraction solution: 80/20 by volume of methanol and water, for mud-like pure methanol (AR) as the extraction solvent alone; HPLC eluent A: water. Eluent B: methanol / acetonitrile 80/20 (v / v). Tween solution: 100ml Tween 20 dissolved in 900ml of water HPLC calibration standard solution: the standard solution dubbed target content 1ug / kg of calibration solution treatment step before the extraction of peanut kernels of similar products (pistachios, almonds, almond shells , peanuts and the like): Weigh 50g syrup-like (accession homogeneous 100-300% water) was added 5gNaCl (for spiked samples added to a dispersion standard solution), add 75ml of pure methanol was then homogenized three minutes or shock extract 30 minutes. For a small amount of the sample, said sample 25g dry uniformly, and 100ml of methanol was added 5g NaCl / water 80/20 (v / v) solution of 3 minutes and then homogenized for 30 minutes to extract or vibration. The extract was filtered with filter paper or centrifugation, the supernatant was transferred to 100ml Erlenmeyer flask. For special peanut butter or other nut butter, its outside layer of fat, it is difficult to be extracted. Said 25g sample was added 5g NaCl, 100ml and 50ml of n-hexane extract solution. Extracted with a homogenizer for 3-5 minutes, the oil phase portion would be soluble in hexane, the supernatant liquid was collected by filtration. Weigh 25g milk cereal type sample in 250ml conical flask, 5g NaCl, 100ml extraction solution (for a standard solution spiked samples were added to a dispersion), and then shaken at 150 rpm on a horizontal shaker for 30 minutes. For milk samples can be weighed and added to the extract dissolved 2-5g, such as poorly soluble can be properly heated and filtered through filter paper to collect the supernatant. Spices, tea, coffee, cocoa, feed, pharmaceuticals, capsicum extract dry sample weighed 20g, 50g mud sample, adding 5g NaCl, 100ml extraction solution, slurry-like add 80ml of pure methanol (standard for spiked samples added to a dispersion solution), and then shaken at 150 rpm on a horizontal shaker for 30 minutes. Filter paper to collect the supernatant. The filtrate was added 20ml of 5ml Tween solution is diluted, dilution with a glass fiber filter. Blank reagent blank value must be determined. Immunoaffinity column (IAC) immunoaffinity column purification will be placed on a solid phase extraction device, a sample corresponding to the second pillar; 10ml PBS solution activated column, the flow rate is maintained at 2 - 3 ml / min, to ensure that the sample before the column reserved small amount (0.5ml) of PBS solution, the activation solution is not collected; 1, peanut extract, milk, nuts and grains analogs: 3.1ml extract was 9.9mlPBS dilution. By dilution with 12.6ml IAC, but can not exceed the flow rate of 5 mL / min. With about 20ml of deionized water rinse pillars, which shall not exceed the flow rate of 10ml 5 mL / min, vacuum 1 minute; 2, spices, tea, coffee, cocoa, feed, pharmaceuticals, capsicum extract: 9ml diluted sample through immunization affinity column at a flow rate of no more than 5 mL / min. With about 20ml of deionized water rinse the column, the flow rate should not exceed 10ml in which 5 mL / min, vacuum 1 minute; eluted with 1.5ml methanol; (the column was connected to a glass apparatus with 0.75ml of methanol twice to the target The eluted and collected in a 15ml centrifuge tube, the initial pressure in the column so that it is probably by 4 drops of liquid droplets and then allowed to naturally flow down by gravity, and finally pass into the air so that all the liquid flows out and collected.) Eluent diluted and mixed with 1.5ml of water, the final volume of 3 ml; after filtration machine test Note: The specific use of immunoaffinity column refer to the instructions 3. Liquid chromatography - fluorescence measuring instrument LC system: Agilent 1200 two yuan Agilent 1200 high-pressure pump vacuum degasser Agilent 1200 Agilent autosampler thermostat device XB-C18 250mm x 4.6 mm 5um flow after 1200 column oven photochemical column derivatization phase: Injection volume: C18,, 4 x52μmmm (acetonitrile: methanol ( 50:50)): water = 35: 65 20ul column temperature: FLD: 1 ml / min 35 ° C excitation: 365nm; emission: 450nm GUODA chromatography Mall Address: Tongbai Road, Zhongyuan District No. 98 Royal Lake Area A Royal Lake Garden Palace Room 811, Building 3103 GUODA chromatography Mall Tel: 0371-68850806 / 68850808 GUODA chromatography Mall Fax: 0371-68811223 / 68811229 GUODA chromatography Mall: GUODA chromatography instrument Mall www.gd226.cn GUODA Chromatography Mall www.guodasepu.com GUODA chromatography Mall mail: 2350470569@qq.com GUODA free 400 Phone: 400-11-58566 GUODA chromatography Mall Online Customer Service QQ: 2350470569